CROSS-REFERENCE TO RELATED APPLICATIONS
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under National Institutes of Health
Grant No. U54CA119335 and National Science Foundation Grant No. CMMI-1031239. The
Government has certain rights in the invention.
FIELD
[0003] The present teachings relate to nanoparticles, drug conjugates, and controlled release
of drug conjugates from the nanoparticles. Methods of making the nanoparticles and
drug conjugates are contemplated.
INTRODUCTION
[0004] Combinatorial drug delivery, or combination therapy, refers to the use of multiple
drugs to treat diseases or disorders in patients such as various cancers. For example,
gemicitabine and paclitaxel are concurrently administered for treating breast cancer;
docetaxel and carboplatin for lung cancer; and doxorubicin and ifosfamide for soft
tissue sarcoma. Combination chemotherapy is usually more effective than individual
chemotherapy as drugs with similar mechanisms act synergistically to enhance therapeutic
efficacy whereas drugs with different mechanisms give cancer cells a higher hurdle
in developing resistance. However, because of the different therapeutic indices, cellular
uptake mechanisms, and
in vivo clearance time among drugs, it is difficult to ensure that the tumors receive the
optimal dosage of each therapeutic agent. Compositions and methods for precisely controlling
the molar ratio among multiple drugs and their concentration taken up by the same
target diseased cells would therefore be beneficial in optimizing combination chemotherapy
regimens.
[0005] Nanoparticulate drug delivery systems have become increasingly attractive in systemic
drug delivery because of their ability to prolong drug circulation half-life, reduce
non-specific uptake, and better accumulate at the tumors through enhanced permeation
and retention (EPR) effect. As a result, several therapeutic nanoparticles such as
Doxil® and Abraxane® are used as the frontline therapies in clinics. But despite the
advancement in nanoparticle drug delivery, most research efforts focus on single drug
encapsulation. Several strategies have been employed to co-encapsulate multiple drugs
into a single nanocarrier, including physical loading into the particle core (see,
e.g.,
X. R. Song, et al. Eur J Pharm Sci 2009, 37, 300-305;
C. E. Soma, et al. Biomaterials 2000, 21, 1-7), chemical conjugation to the particle surface (
see, e.g., L. Zhang, et al. ChemMedChem 2007, 2, 1268-1271), and covalent linkage to the polymer backbone prior to nanoparticle synthesis (
see, e.g., T. Lammers, et al. Biomaterials 2009, 30, 3466-3475;
Y. Bae, et al. J Control Release 2007, 122, 324-330;
N. Kolishetti, et al. Proc Natl Acad Sci U S A 2010, 107, 17939-17944). However, controlling the ratios of different types of drugs in the same nanoparticles
remains a major challenge because of factors such as steric hindrance between the
different drug molecules and the polymer backbones, batch-to-batch heterogeneity in
conjugation chemistry, and variability in drug-to-drug and drug-to-polymer interactions.
[0006] Many pharmaceutically active agents possess multiple functional groups that are readily
modified chemically. Several prodrugs have been synthesized based on these functional
groups. For instance, gemcitabine has been acylated through its primary amine to improve
its stability in blood; paclitaxel has been pegylated through its hydroxyl groups
to improve its water solubility; and doxorubicin has been conjugated to polymers through
hydrazone linkage to its ketonic group for nanoparticle encapsulation. It has been
demonstrated that modifications through the aforementioned functional groups do not
reduce the therapeutic efficacy of chemotherapy drugs as the modified drugs either
retain their chemical activities or release the drug content intracellularly through
pH- or enzyme-sensitive response.
[0007] Therefore, what is needed are compositions comprising ratiometrically controlled
drug combinations, methods of synthesizing such ratiometric compositions, and combination
therapy methods of using such compositions.
SUMMARY
[0008] The invention relates to a method for nanoencapsulation of a plurality of drugs comprising:
separately linking each of the plurality of drugs with a corresponding polymer backbone
with nearly 100% loading efficiency by forming the corresponding polymer backbone
by ring opening polymerization beginning with the corresponding drug, wherein each
of the corresponding polymer backbones has the same or similar physicochemical properties
and has approximately the same chain length;
mixing the plurality of linked drugs and polymers at selectively predetermined ratios
at selectively and precisely controlled drug ratios; and
synthesizing the mixed plurality of linked drugs and polymers into a nanoparticle.
[0009] The present teachings include ratiometric combinatorial drug delivery including nanoparticles,
multi-drug conjugates, pharmaceutical compositions and methods of producing such compositions.
In one embodiment, a nanoparticle is provided that includes an inner sphere and an
outer surface, the inner sphere containing a combination of conjugated drugs connected
by a stimuli-sensitive bond and having a predetermined ratio, wherein the conjugated
drugs have the following formula:
(X-Y-Z)
n
wherein X is a pharmaceutically active agent, Y is a stimuli-sensitive linker, and
Z is not X, and is a pharmaceutically active agent or hydrogen. In various aspects,
n is an integer greater than or equal to 2. In another aspect, each individual conjugated
drug of the combination comprises a predetermined molar weight percentage from about
1% to about 99%, provided that the sum of all individual conjugated drug molar weight
percentages of the combination is 100%. In various aspects of the present embodiment,
about 100% of drugs contained in the inner sphere are conjugated.
[0010] In various aspects, X can independently be an antibiotic, antimicrobial, growth factor,
chemotherapeutic agent, and combinations thereof. For instance, X can independently
include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, and pharmaceutically acceptable salts thereof.
In various aspects, Z can independently be an antibiotic, antimicrobial, growth factor,
chemotherapeutic agent, hydrogen, and combinations thereof. For instance, Z can independently
include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, pharmaceutically acceptable salts thereof,
and hydrogen.
[0011] In various aspects, Y is a pH-sensitive linker. For instance, Y can include C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0012] In various aspects, the outer surface of the nanoparticle can include a cationic
or anionic functional group.
[0013] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula I:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W' is phenyl or tert-butyl oxy; and 'R' is hydrogen or alkyl. For instance,
'p' can be 3; 'X' can be chloride; 'W' can be phenyl and 'R' can be hydrogen.
[0014] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula II:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W
1' and 'W
2' are independently selected from phenyl or tert-butyl oxy; and 'R' is hydrogen or
alkyl. For instance, 'p' can be 3; 'X' is chloride; 'W
1' and 'W
2' can be phenyl and 'R' can be hydrogen.
[0015] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula III:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; and 'W' is slected from phenyl or tert-butyl oxy. For instance, 'p' can be 3;
and 'W' can be phenyl.
[0016] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula IV:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy; and 'V
1' and 'V
2' are independently selected from -CH
3 or -CH
2OH. For instance,'W' can be phenyl; and 'V
1' and 'V
2' can be -CH
2OH.
[0017] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula V:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy. For instance, 'W' can be phenyl.
[0018] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula VI:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0019] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula VII:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0020] In various aspects, the nanoparticle is about 10 nm to about 10 µm in diameter, and
in certain aspects about 30 nm to about 300 nm in diameter.
[0021] In another embodiment, a multi-drug conjugate is provided having the following formula:
X-Y-Z
wherein X and Z are pharmaceutically active agents independently selected from the
group consisting of an antibiotic, antimicrobial, growth factor, and chemotherapeutic
agent; and Y is a stimuli-sensitive linker, wherein the conjugate releases at least
one pharmaceutically active agent upon delivery of the conjugate to a target cell.
[0022] In various aspects of the present embodiment, Y is a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof. For instance, Y can be a C
3 straight chain alkyl or a ketone. In various aspects, the pharmaceutically active
agent comprises an anticancer chemotherapy agent. For instance, X and Y can independently
be doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, or pharmaceutically acceptable salts thereof.
[0023] In yet another aspect, the conjugate has Formula I:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W' is phenyl or tert-butyl oxy; and 'R' is hydrogen or alkyl. For instance,
'p' can be 3; 'X' can be chloride; 'W' can be phenyl and 'R' can be hydrogen.
[0024] In another aspect, the conjugate has Formula II:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W
1' and 'W
2' are independently selected from phenyl or tert-butyl oxy; and 'R' is hydrogen or
alkyl. For instance, 'p' can be 3; 'X' can be chloride; 'W
1' and 'W
2' can be phenyl and 'R' can be hydrogen.
[0025] In another aspect, the conjugate has Formula III:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; and 'W' is slected from phenyl or tert-butyl oxy. For instance, 'p' can be 3;
and 'W' can be phenyl.
[0026] In another aspect, the conjugate has Formula IV:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy; and 'V
1' and 'V
2' are independently selected from -CH
3 or -CH
2OH. For instance, 'W' can be phenyl; and 'V
1' and 'V
2' can be --CH
2OH.
[0027] In another aspect, the conjugate has Formula V:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy. For instance, 'W' can be phenyl.
[0028] In another aspect, the conjugate has Formula VI:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0029] In another aspect, the conjugate has Formula VII:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0030] In yet another embodiment, a multi-drug conjugate is provided comprising a pharmaceutically
active agent covalently bound to a plurality of stimuli-sensitive linkers, wherein
each linker is covalently bound to at least one additional pharmaceutically active
agent, wherein the conjugate releases at least one pharmaceutically active agent upon
delivery to a target cell. In one aspect, the stimuli-sensitive linker can be a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, or combinations thereof. For instance, the linker can be a
C
3 straight chain alkyl. In yet another instance, the linker can comprise a ketone.
[0031] In yet another aspect, the pharmaceutically active agent comprises anticancer chemotherapy
agents. For instance, the pharmaceutically active agent can include doxorubicin, camptothecin,
gemicitabine, carboplatin, oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin,
aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin,
5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,
etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine,
estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte, 4-desacetylvinblastine,
epothilone B, docetaxel, maytansanol, epothilone A, combretastatin, pharmaceutically
active analogs thereof, and pharmaceutically acceptable salts thereof.
[0032] In another embodiment, a pharmaceutical composition is provided comprising the multi-drug
conjugate above, or a pharmaceutically acceptable salt thereof, in a pharmaceutically
acceptable vehicle.
[0033] In yet another embodiment, a method is provided for controlling ratios of conjugated
drugs contained in a nanoparticle inner sphere, the method comprising: a) synthesizing
a combination of a first drug independently conjugated to a stimuli-sensitive linker,
and a second drug independently conjugated to a linker having the same composition,
wherein the first drug conjugate and second drug conjugate have a predetermined ratio;
b) adding the combination to an agitated solution comprising a polar lipid; and c)
adding water to the agitated solution, wherein nanoparticles are produced having a
controlled ratio of conjugated drugs contained in the inner sphere. In various aspects
of the present embodiment, about 100% of drugs contained in the inner sphere are conjugated.
[0034] In one aspect, the first drug and the second drug can independently include an antibiotic,
antimicrobial, antiviral, growth factor, chemotherapeutic agent, and combinations
thereof. For instance, the first drug and the second drug are independently selected
from the group consisting of doxorubicin, camptothecin, gemicitabine, carboplatin,
oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate,
methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0035] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the stimuli-sensitive linker is selected from the group consisting of C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0036] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional drug independently
conjugated to a stimuli-sensitive linker having the same composition.
[0037] In yet another embodiment, a method is provided for controlling ratios of conjugated
drugs contained in a nanoparticle inner sphere, the method comprising: a) synthesizing
a combination of (i) a first drug and a second drug conjugated by a first stimuli-sensitive
linker, and (ii) a first drug and a second drug conjugated by a second stimuli-sensitive
linker, wherein the first drug conjugate and second drug conjugate have a predetermined
ratio; b) adding the combination to an agitated solution comprising a polar lipid;
and c) adding water to the agitated solution, wherein nanoparticles are produced having
a controlled ratio of conjugated drugs contained in the inner sphere. In various aspects
of the present embodiment, about 100% of drugs contained in the inner sphere are conjugated.
[0038] In one aspect, the first drug and the second drug are independently selected from
the group consisting of an antibiotic, antimicrobial, antiviral, growth factor, chemotherapeutic
agent, and combinations thereof. For instance, the first drug and the second drug
can independently include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin,
epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0039] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the first stimuli-sensitive linker and the second stimuli-sensitive linker can independently
include C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0040] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional conjugate of
a first drug and a second drug conjugated by a stimuli-sensitive linker other than
those present in the combination.
[0041] In another embodiment, a method is provided for producing a combination of conjugated
drugs having a predetermined ratio in a nanoparticle, said nanoparticle comprising
an inner sphere, the method comprising: a) adding to an agitated solution comprising
a polar lipid a combination of a first drug independently conjugated to a stimuli-sensitive
linker, and a second drug independently conjugated to a linker having the same composition,
wherein the first drug conjugate and the second drug conjugate have a predetermined
ratio; and b) adding water to the agitated solution, wherein nanoparticles are produced
containing in the inner sphere the conjugated drugs having a predetermined ratio.
In various aspects, the method can further comprise: c) isolating nanoparticles having
a diameter less than about 300 nm. In various aspects of the present embodiment, about
100% of drugs contained in the inner sphere are conjugated.
[0042] In various aspects, the first drug and the second drug are independently selected
from the group consisting of an antibiotic, antimicrobial, growth factor, chemotherapeutic
agent, and combinations thereof. For instance, the first drug and the second drug
can independently include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin,
epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0043] In yet another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For
instance, the stimuli-sensitive linker can be C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, or combinations thereof.
[0044] In yet another aspect, the combination of conjugated drugs having a predetermined
ratio further comprise a third drug independently conjugated to a stimuli-sensitive
linker having the same composition. In various aspects, the solution comprising a
polar lipid further comprises a functionlalized polar lipid.
[0045] In yet another embodiment, a method is provided for producing a combination of conjugated
drugs having a predetermined ratio in a nanoparticle, said nanoparticle comprising
an inner sphere, the method comprising: a) adding to an agitated solution comprising
a polar lipid a combination of (i) a first drug and second drug conjugated by a first
stimuli-sensitive linker, and (ii) a first drug and a second drug conjugated by a
second stimuli-sensitive linker, wherein the first drug conjugate and second drug
conjugate have a predetermined ratio; and b) adding water to the agitated solution,
wherein nanoparticles are produced containing in the inner sphere the conjugated drugs
having a predetermined ratio. In various aspects, the method can further comprise:
c) isolating nanoparticles having a diameter less than about 300 nm. In various aspects
of the present embodiment, about 100% of drugs contained in the inner sphere are conjugated.
[0046] In one aspect, the first drug and the second drug can independently include an antibiotic,
antimicrobial, growth factor, chemotherapeutic agent, and combinations thereof. For
instance, the first drug and the second drug are independently selected from the group
consisting of doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, and pharmaceutically acceptable salts thereof.
[0047] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the first stimuli-sensitive linker and the second stimuli-sensitive linker can independently
be C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0048] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional conjugate of
a first drug and a second drug conjugated by a stimuli-sensitive linker other than
those present in the combination. In various aspects, the solution comprising a polar
lipid further comprises a functionlalized polar lipid.
[0049] In yet another disclosure, a method is provided for treating a disease or condition,
the method comprising administering a therapeutically effective amount of the nanoparticle
above to a subject in need thereof. In one aspect, the disease is a proliferative
disease including lymphoma, renal cell carcinoma, prostate cancer, lung cancer, pancreatic
cancer, melanoma, colorectal cancer, ovarian cancer, breast cancer, glioblastoma multiforme
and leptomeningeal carcinomatosis. In another aspect, the disease is a heart disease
including Atherosclerosis, Ischemic heart disease, Rheumatic heart disease, Hypertensive
heart disease, Infective endocarditis, Coronary heart disease, and Constrictive pericarditis.
In another aspect, the disease is an ocular disease selected from the group consisting
of macular edema, retinal ischemia, macular degeneration, uveitis, blepharitis, keratitis,
rubeosis iritis, iridocyclitis, conjunctivitis, and vasculitis. In another aspect,
the disease is a lung disease including asthma, Chronic Bronchitis, Cystic Fibrosis,
Emphysema, Pneumonia, lung cancer, Primary Pulmonary Hypertension, Pulmonary Arterial
Hypertension, and Tuberculosis. In yet another aspect, the disease includes bacterial
infection, viral infection, fungal infection, and parasitic infection.
[0050] In various aspects of the present disclosure, the nanoparticle is administered systemically.
In another aspect, the nanoparticle is administered locally. In yet another aspect,
the local administration is via implantable metronomic infusion pump.
[0051] In yet another disclosure, a method is provided for treating a disease or condition,
the method comprising administering a therapeutically effective amount of the multi-drug
conjugate above to a subject in need thereof. In one aspect, the disease is a proliferative
disease including lymphoma, renal cell carcinoma, prostate cancer, lung cancer, pancreatic
cancer, melanoma, colorectal cancer, ovarian cancer, breast cancer, glioblastoma multiforme
and leptomeningeal carcinomatosis. In one aspect, the disease is a heart disease including
Atherosclerosis, Ischemic heart disease, Rheumatic heart disease, Hypertensive heart
disease, Infective endocarditis, Coronary heart disease, and Constrictive pericarditis.
In one aspect, the disease is an ocular disease including macular edema, retinal ischemia,
macular degeneration, uveitis, blepharitis, keratitis, rubeosis iritis, iridocyclitis,
conjunctivitis, and vasculitis. In one aspect, the disease is a lung disease including
asthma, Chronic Bronchitis, Cystic Fibrosis, Emphysema, Pneumonia, lung cancer, Primary
Pulmonary Hypertension, Pulmonary Arterial Hypertension, and Tuberculosis. In yet
another aspect, the disease is selected from the group consisting of bacterial infection,
viral infection, fungal infection, and parasitic infection.
[0052] In various aspects of the present disclosure, the multi-drug conjugate is administered
systemically. In another aspect, the multi-drug conjugate is administered locally.
In yet another aspect, the local administration is via implantable metronomic infusion
pump.
[0053] In yet another disclosure, a method is provided for sequentially delivering a drug
conjugate to a target cell, the method comprising administering a nanoparticle above
to the target cell and triggering multi-drug conjugate release. In various aspects
of the present embodiment, the nanoparticle is administered systemically. In another
aspect, the nanoparticle is administered locally. In yet another aspect, the local
administration is via implantable metronomic infusion pump.
[0054] In yet another embodiment, a method is provided for nanoencapsulation of a plurality
of drugs comprising separately linking each of the plurality of drugs with a corresponding
polymer backbone with nearly 100% loading efficiency by forming the corresponding
polymer backbone by ring opening polymerization beginning with the corresponding drug,
wherein each of the corresponding polymer backbones has the same or similar physicochemical
properties and has approximately the same chain length; mixing the plurality of linked
drugs and polymers at selectively predetermined ratios at selectively and precisely
controlled drug ratios; and synthesizing the mixed plurality of linked drugs and polymers
into a nanoparticle.
[0055] In various aspects, the plurality of drugs can independently include an antibiotic,
antimicrobial, growth factor, chemotherapeutic agent, and combinations thereof. For
instance, the plurality of drugs can independently include doxorubicin, camptothecin,
gemicitabine, carboplatin, oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin,
aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin,
5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,
etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine,
estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte, 4-desacetylvinblastine,
epothilone B, docetaxel, maytansanol, epothilone A, combretastatin, pharmaceutically
active analogs thereof, and pharmaceutically acceptable salts thereof.
[0056] In various aspects, the polymer backbone is a stimuli-sensitive linker. For instance,
the stimuli-sensitive linker can include a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0057] These and other features, aspects and advantages of the present teachings will become
better understood with reference to the following description, examples and appended
claims.
DRAWINGS
[0058] Those of skill in the art will understand that the drawings, described below, are
for illustrative purposes only. The drawings are not intended to limit the scope of
the present teachings in any way.
Figure 1. Schematic illustration of a dual-drug loaded lipid-polymer hybrid nanoparticle,
of which the polymeric core consists of two distinct drug-polymer conjugates with
ratiometric control over drug loading.
Figure 2. Chemical characterization of the drug-polymer conjugates. (A) Schematic
description of the living ring-opening polymerization of 1-lactide catalyzed by an
activated metal alkoxide complex. (B) Qualitative 1H-NMR spectra showing the characteristic proton resonance peaks of DOX-PLA (upper
panel) and CPT-PLA (lower panel). (C) Gel permeation chromatograms of DOX-PLA (red
dashed line) and CPT-PLA (black solid line).
Figure 3. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) measurements
showing the morphology and size of lipid-polymer hybrid nanoparticles with the polymer
cores consisting of: (A) DOX-PLA conjugates, (B) CPT-PLA conjugates, or (C) DOX-PLA
and CPT-PLA conjugates with a molar ratio of 1:1.
Figure 4. Quantification of DOX and CPT loading efficiency in dual-drug loaded nanoparticles
(containing both DOX-PLA and CAP-PLA) and single-drug loaded nanoparticles (containing
DOX-PLA or CPT-PLA), respectively. NPs: nanoparticles.
Figure 5. Cellular colocalization and cytotoxicity studies of the DOX-PLA and CPT-PLA
loaded dual-drug nanoparticles. (A) Fluorescence microscopy images showing the colocalization
of DOX and CPT in the cellular compartment of MDB-MB-435 breast cancer cells. (B)
A comparative study of cellular cytotoxicity of the DOX-PLA and CPT-PLA loaded dual-drug
nanoparticles against the MDB-MB-435 breast cancer cells. The ratios shown in figure
legends are the molar ratios of DOX-PLA to CPT-PLA. Solid lines represent the dual-drug
loaded nanoparticles and dashed lines represent the cocktail mixture of DOX-PLA loaded
and CPT-PLA loaded single-drug nanoparticles. All samples were incubated with cells
for 24 h, and the cells were subsequently washed and incubated in media for a total
of 72 h prior to MTT assay (n=4).
Figure 6. Mass spectrum (ESI-positive ion mode) of 2-((2,6-diisopropylphenyl)amido)-4-((2,6diisopropylphenyl)-imino)-2-pentene
(BDI).
Figure 7. 1H-NMR characterization of 2-((2,6-diisopropylphenyl)amido)-4-((2,6diisopropylphenyl)
-imino)-2-pentene (BDI).
Figure 8. 1H-NMR characterization of (BDI)ZnN(SiMe3)2 complex catalyst.
Figure 9. Synthesis scheme of paclitaxel (PTXL) and gemcitabine hydrochloride (GEM)
conjugate (PTXL-GEM conjugate, compound 2).
Figure 10. Characterization of PTXL-GEM conjugates using (A) 1H-NMR spectroscopy showing the characteristic protons, and (B) high resolution mass spectrum determining the exact mass and corresponding molecular
formula of the drug conjugates.
Figure 11. Hydrolysis and cellular cytotoxicity of PTXL-GEM conjugates. (A) HPLC chromatograms
of PTXL-GEM conjugates (a) before and (b) after 24 hrs of incubation in water/acetonitrile (75/25, v/v) solution at pH = 7.4.
(B) Hydrolysis kinetics of PTXL-GEM conjugates at pH = 6.0 and pH = 7.4. (C) Time dependent
comparative cytotoxicity of PTXL-GEM conjugates with the corresponding mixture of
free PTXL and free GEM drugs at 100 nM concentration against XPA3 human pancreatic
cancer cell line (n = 8).
Figure 12. Characterization of PTXL-GEM conjugates loaded lipid-coated polymeric nanoparticles
(NPs). (A) Schematic illustration of a PTXL-GEM conjugates loaded nanoparticle. (B) Representative scanning electron microscopy (SEM) image of PTXL-GEM conjugates loaded
nanoparticles. (C) Diameter and surface zeta-potential of PTXL-GEM conjugates loaded nanoparticles and
empty nanoparticles measured by dyanamic light scattering (DLS).
Figure 13. (A) PTXL-GEM conjugates loading yield at various initial weight ratios of PTXL-GEM conjugates/excipient
(PLGA polymer). (B) Cellular cytotoxicity of PTXL-GEM conjugates loaded nanoparticles and free PTXL-GEM
conjugates (compound 2) at various drug conjugate concentrations against XPA3 human
pancreatic cancer cell line. All samples were incubated with cells for 24 hrs, and
the cells were subsequently washed and incubated in media for a total of 72 hrs before
assessing cell viability in each group (n = 8).
Figure 14. 1H NMR spectrum of paclitaxel.
Figure 15. 1H NMR spectrum of compound 1.
Figure 16. ESI-MS (positive) mass spectrum of compound 1.
Figure 17. ESI-MS (positive) mass spectrum of paclitaxel recovered from the hydrolyzed
PTXL-GEM conjugates with an HPLC retention time of 6.2 min.
Figure 18. ESI-MS (positive) mass spectrum of gemcitabine recovered from the hydrolyzed
PTXL-GEM conjugates with an HPLC retention time of 1.8 min.
Figure 19. Synthesis scheme of paclitaxel (Ptxl) and cisplatin conjugate (Ptxl-Pt(IV)
conjugate) as a representative hydrophobic-hydrophilic drug conjugate.
Figure 20. Characterization of Ptxl-Pt(IV) conjugate using (A) 1H-NMR spectroscopy showing the characteristic protons, and (B) high resolution mass
spectrum determining the exact mass and corresponding molecular formula of the Ptxl-Pt(IV)
conjugate.
Figure 21. Characterization of Ptxl-Pt(IV) conjugates loaded nanoparticles. (A) Schematic
illustration of Ptxl-Pt(IV) conjugates loaded lipid coated polymeric nanoparticles.
(B) Dynamic light scattering (DLS) measurement of Ptxl-Pt(IV) loaded nanoparticles.
(C) Representative scanning electron microscopy (SEM) image of Ptxl-Pt(IV) loaded
nanoparticles. Inset: high-resolution SEM image of Ptxl-Pt(IV) loaded nanoparticles
Figure 22. (A) Cellular cytotoxicity of free Ptxl-Pt(IV) conjugates and Ptxl-Pt(IV)
conjugates loaded nanoparticles (NPs) at various drug concentration against A2780
human ovarian cancer cell line. All samples were incubated with cells for 24 hrs,
and the cells were subsequently washed and incubated in fresh media for a total of
72 hrs before cell viability using the ATP assay (n=8). (B,C) Representative phase
contrast microscopy images of A2780 cells treated with (B) free Ptxl-Pt(IV) drug conjugates
and (C) Ptxl-Pt(IV) conjugates loaded nanoparticles, respectively, at a drug concentration
of 300 nM.
Figure 23. 1H NMR spectrum of cis-trans-cis PtCl2(OCOCH2CH2CH2COOH)2(NH3)2.
Figure 24. Drug loading yield of PTXL conjugates.
DETAILED DESCRIPTION
Abbreviations and Definitions
[0059] To facilitate understanding of the invention, a number of terms and abbreviations
as used herein are defined below as follows:
[0060] When introducing elements of the present invention or the preferred embodiment(s)
thereof, the articles "a", "an", "the" and "said" are intended to mean that there
are one or more of the elements. The terms "comprising", "including" and "having"
are intended to be inclusive and mean that there may be additional elements other
than the listed elements.
[0061] The term "and/or" when used in a list of two or more items, means that any one of
the listed items can be employed by itself or in combination with any one or more
of the listed items. For example, the expression "A and/or B" is intended to mean
either or both of A and B,
i.e. A alone, B alone or A and B in combination. The expression "A, B and/or C" is intended
to mean A alone, B alone, C alone, A and B in combination, A and C in combination,
B and C in combination or A, B, and C in combination.
[0062] In the descriptions of molecules and substituents, molecular descriptors can be combined
to produce words or phrases that describe substituents. Such descriptors are used
in this document. Examples include such terms as aralkyl (or arylalkyl), heteroaralkyl,
heterocycloalkyl, cycloalkylalkyl, aralkoxyalkoxycarbonyl and the like. A specific
example of a compound encompassed with the latter descriptor aralkoxyalkoxycarbonyl
is C
6H
5-CH
2-CH
2-O-CH
2-O-C(O) wherein C
6H
5 is phenyl. It is also to be noted that a substituents can have more than one descriptive
word or phrase in the art, for example, heteroaryloxyalkylcarbonyl can also be termed
heteroaryloxyalkanoyl. Such combinations are used herein in the description of the
compounds and methods of this invention and further examples are described herein.
[0063] Alkyl: The term "alkyl" as used herein describes substituents which are preferably
lower alkyl containing from one to eight carbon atoms in the principal chain and up
to about 20 carbon atoms. The principal chain may be straight or branched chain or
cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.
[0064] Analog: The term "analog" as used herein may refer to a compound in which one or
more atoms are replaced with a different atom or group of atoms. The term may also
refer to compounds with an identity of atoms but of different isomeric configuration.
Such isomers may be constitutional isomers,
i.e. structural isomers having different bonding arrangements of their atoms or stereoisomers
having identical bonding arrangements but different spatial arrangements of the constituent
atoms.
[0065] Anionic: The term "anionic" as used herein refers to substances capable of forming
ions in aqueous media with a net negative charge.
[0066] Anionic functional group: The term "anionic functional group" as used herein refers
to functional group as defined herein which possesses a net negative charge. Representative
anionic functional groups include carboxylic, sulfonic, phosphonic, their alkylated
derivatives, and so on.
[0067] Cationic: The term "cationic" as used herein refers to substances capable of forming
ions in aqueous media with a net positive charge.
[0068] Functional group: The term "functional group" as used herein, refers to a chemical
group that imparts a particular function to an article (
e.g., nanoparticle) bearing the chemical group. For example, functional groups can include
substances such as antibodies, oligonucleotides, biotin, or streptavidin that are
known to bind particular molecules; or small chemical groups such as amines, carboxylates,
and the like.
[0069] Halogen: The terms "halogen" or "halo" as used herein, alone or as part of a group
of atoms, refer to chlorine, bromine, fluorine, and iodine.
[0070] Nanoparticle: The term "nanoparticle" as used herein refers to unilamellar or multilamellar
lipid vesicles which enclose a fluid space and has a diameter of between about 1 nm
and about 1000 nm. Similarly, by the term "nanoparticles" is meant a plurality of
particles having an average diameter of between about 1 nm and about 1000 nm. The
term can also include vesicles as large as 10,000 nm depending on the environment
such nanoparticles are administered to a subject, for example, locally to a tumor
in situ via implantable pump or via syringe. For systemic use, an average diameter of about
30 nm to about 300 nm is preferred. The walls of the vesicles, also referred to as
a membrane, are formed by a bimolecular layer of one or more lipid components (
e.
g., multiple phospholipids and cholesterol) having polar heads and non-polar tails,
such as a phospholipid. In an aqueous (or polar) solution, and in a unilamellar nanoparticle,
the polar heads of one layer orient outwardly to extend into the surrounding medium,
and the non-polar tail portions of the lipids associate with each other, thus providing
a polar surface and a non-polar core in the wall of the vesicle. In a multilamellar
nanoparticle, the polar surface of the vesicle also extends to the core of the liposome
and the wall is a bilayer. The wall of the vesicle in either of the unilamellar or
multilamellar nanoparticles can be saturated or unsaturated with other lipid components,
such as cholesterol, free fatty acids, and phospholipids. In such cases, an excess
amount of the other lipid component can be added to the vesicle wall which will shed
until the concentration in the vesicle wall reaches equilibrium, which can be dependent
upon the nanoparticle environment. Nanoparticles may also comprise other agents that
may or may not increase an activity of the nanoparticle. For example, polyethylene
glycol (PEG) can be added to the outer surface of the membrane to enhance bioavailability.
In other examples, functional groups such as antibodies and aptamers can be added
to the outer surface of the membrane to enhance site targeting, such as to cell surface
epitopes found in cancer cells. The membrane of the nanoparticles can also comprise
particles that can be biodegradable, cationic nanoparticles including, but not limited
to, gold, silver, and synthetic nanoparticles. An example of a biocompatible synthetic
nanoparticle includes polystyrene and the like.
[0071] Pharmaceutically active: The terms "pharmaceutically active" as used herein refer
to the beneficial biological activity of a substance on living matter and, in particular,
on cells and tissues of the human body. A "pharmaceutically active agent" or "drug"
is a substance that is pharmaceutically active and a "pharmaceutically active ingredient"
(API) is the pharmaceutically active substance in a drug.
[0072] Pharmaceutically acceptable: The terms "pharmaceutically acceptable" as used herein
means approved by a regulatory agency of the Federal or a state government or listed
in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to
other formulations that are safe for use in animals, and more particularly in humans
and/or non-human mammals.
[0073] Pharmaceutically acceptable salt: The terms "pharmaceutically acceptable salt" as
used herein refer to acid addition salts or base addition salts of the compounds,
such as the multi-drug conjugates, in the present disclosure. A pharmaceutically acceptable
salt is any salt which retains the activity of the parent compound and does not impart
any deleterious or undesirable effect on a subject to whom it is administered and
in the context in which it is administered. Pharmaceutically acceptable salts include,
but are not limited to, metal complexes and salts of both inorganic and carboxylic
acids. Pharmaceutically acceptable salts also include metal salts such as aluminum,
calcium, iron, magnesium, manganese and complex salts. In addition, pharmaceutically
acceptable salts include, but are not limited to, acid salts such as acetic, aspartic,
alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic, bisulfuric,
bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, citric, edetic,
edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic,
glycolylarsanilic, hexamic, hexylresorcjnoic, hydrabamic, hydrobromic, hydrochloric,
hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic, malonic,
mandelic, methanesulfonic, methylnitric, methylsulfuric, mucic, muconic, napsylic,
nitric, oxalic, p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogen
phosphoric, dihydrogen phosphoric, phthalic, polygalactouronic, propionic, salicylic,
stearic, succinic, sulfamic, sulfanlic, sulfonic, sulfuric, tannic, tartaric, teoclic,
toluenesulfonic, and the like. Pharmaceutically acceptable salts may be derived from
amino acids including, but not limited to, cysteine. Methods for producing compounds
as salts are known to those of skill in the art (see, for example,
Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH;
Verlag Helvetica Chimica Acta, Zürich, 2002;
Berge et al., J Pharm. Sci. 66: 1, 1977).
[0074] Pharmaceutically acceptable carrier: The terms "pharmaceutically acceptable carrier"
as used herein refers to an excipient, diluent, preservative, solubilizer, emulsifier,
adjuvant, and/or vehicle with which a compound, such as a multi-drug conjugate, is
administered. Such carriers may be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents. Water is a preferred carrier when a compound is
administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions
may also be employed as liquid carriers, particularly for injectable solutions. Suitable
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A compound,
if desired, may also combine minor amounts of wetting or emulsifying agents, or pH
buffering agents such as acetates, citrates or phosphates. Antibacterial agents such
as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for
the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier.
Methods for producing compounds in combination with carriers are known to those of
skill in the art.
[0075] Phospholipid: The term "phospholipid", as used herein, refers to any of numerous
lipids contain a diglyceride, a phosphate group, and a simple organic molecule such
as choline. Examples of phospholipids include, but are not limited to, Phosphatidic
acid (phosphatidate) (PA), Phosphatidylethanolamine (cephalin) (PE), Phosphatidylcholine
(lecithin) (PC), Phosphatidylserine (PS), and Phosphoinositides which include, but
are not limited to, Phosphatidylinositol (PI), Phosphatidylinositol phosphate (PIP),
Phosphatidylinositol bisphosphate (PIP2) and Phosphatidylinositol triphosphate (PIP3).
Additional examples of PC include DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DRPC,
and DEPC as defined in the art.
[0076] Stimuli-Sensitive Linker: As used herein, the term "stimuli-sensitive linker" refers
to a carbon chain that can contain heteroatoms (
e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long.
Stimuli-sensitive linkers may be substituted with various substituents including,
but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino,
trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic,
cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol,
and ureido groups. Those of skill in the art will recognize that each of these groups
may in turn be substituted. Examples of stimuli-sensitive linkers include, but are
not limited to, pH sensitive linkers, protease cleavable peptide linkers, nuclease
sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive
carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile
linkers, enzyme cleavable linkers (
e.
g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavable linkers,
and so forth.
[0077] Substituted: The term "substituted" as used herein refers to one or more substitutions
that are common in the art. The terms "optionally substituted" means that a group
may be unsubstituted or substituted with one or more substituents. Suitable substituents
for any of the groups defined above may include moieties such as alkyl, cycloalkyl,
alkenyl, alkylidenyl, aryl, heteroaryl, heterocyclyl, halo (e.g., chloro, bromo, iodo
and fluoro), cyano, hydroxy, alkoxyl, aroxyl, sulfhydryl (mercapto), alkylthio, arylthio,
amino, substituted amino, nitro, carbamyl, keto (oxo), acyl, glycolyl, glycyl, hydrazino,
guanyl, sulfamyl, sulfonyl, sulfinyl, thioalkyl-C(O)--, thioalkyl-CO
2--, and the like.
[0078] Therapeutically Effective Amount: As used herein, the term "therapeutically effective
amount" refers to those amounts that, when administered to a particular subject in
view of the nature and severity of that subject's disease or condition, will have
a desired therapeutic effect,
e.g., an amount which will cure, prevent, inhibit, or at least partially arrest or partially
prevent a target disease or condition. More specific embodiments are included in the
Pharmaceutical Preparations and Methods of Administration section below.
Ratiometric Combinatorial Drug Delivery
[0079] The present teachings include ratiometric combinatorial drug delivery including nanoparticles,
multi-drug conjugates, pharmaceutical compositions, methods of producing such compositions
and methods of using such compositions, including in the treatment of diseases and
conditions using drug combinations.
[0080] A combinatorial drug conjugation approach is provided to enable multi-drug delivery.
In one example, hydrophobic and hydrophilic drugs were covalently conjugated using
a hydrolysable linker and then encapsulated into lipid-polymer hybrid nanoparticles
for combined delivery. In one non-limiting example, the ratio between two drugs co-delivered,
some with drastically different properties, included various ratios including a 1:1
drug-drug ratio, and in other examples 3:1 and 1:3 ratios. As disclosed herein, such
ratios can be controlled by the different molar amounts of the drugs in combination
which results in versatile multi-drug encapsulation schemes.
[0081] In one aspect, each different drug molecule is linked to an individual linker backbone
that has the same physicochemical properties and nearly the same chain length (
i.e. a drug-linker). These drug-linker conjugates can be subsequently mixed at predetermined
ratios prior to or during nanoparticle synthesis. The long and sharply distributed
linker, in some examples a polymer chain, can provide each drug molecule a predominant
and uniform hydrophobic property, and yield near 100% drug loading efficiency upon
nanoparticle formation. In various aspects, the linkers can be stimuli-sensitive such
that the linked drug is cleaved upon a change in the nanoparticle or multi-drug conjugate
environment, such as a difference in pH.
[0082] In another aspect, an individual drug molecule is linked to another individual drug
molecule, each being linked through different linkers. These drug-drug conjugates
can be subsequently mixed or created at predetermined ratios prior to or during nanoparticle
synthesis. The hydrophobic properties of these conjugates can be different and the
linkers can have different stimuli-sensitive activities. This can result in sequential
drug delivery as one linker can be cleaved to release a drug at a certain environmental
state, and a second linker can release the same or different drug upon a change in
environmental state, such as a different pH.
[0083] As provided in one non-limiting example, the synthesis of a drug-linker conjugate
with two different pharmaceutically active agents, doxorubicin (DOX) and camptothecin
(CPT), is provided. Utilizing ring-opening polymerization of 1-lactide, DOX and CPT
polymer conjugates were synthesized using metal-amido catalyst, which reacts selectively
with hydroxyl groups of the drug molecules to initiate polymerization (
R. Tong, J. Cheng, Angew Chem Int Ed Engl 2008, 47, 4830-4834;
R. Tong, J. Cheng, Angew Chem 2008, 120, 4908-4912;
R. Tong, J. Cheng, Bioconjug Chem 2010, 21, 111-121;
R. Tong, J. Cheng, J Am Chem Soc 2009, 131, 4744-4754). Using a nanoprecipitation technique (Figure 1), the drug-polymer conjugates were
quantitatively loaded into lipid-polymer hybrid nanoparticles at high loading efficiency
and precisely controlled drug ratios.
See B. M. Chamberlain, et al. J Am Chem Soc 2001, 123, 3229-3238;
L. Zhang, et al. ACS Nano 2008, 2, 1696-1702. The combinatorial treatment provided herein shows superior efficacy to cocktail
therapy
in vitro and offers a solution to the aforementioned limitations in multi-drug encapsulation
into the same nanoparticles.
Ratiometrically Controlled Nanoparticles
[0084] Therefore, in one embodiment, a nanoparticle is provided that includes an inner sphere
and an outer surface, the inner sphere containing a combination of conjugated drugs
connected by a stimuli-sensitive bond and having a predetermined ratio, wherein the
conjugated drugs have the following formula:
(X-Y-Z)
n
wherein X is a pharmaceutically active agent, Y is a stimuli-sensitive linker, and
Z is not X, and Z is a pharmaceutically active agent or hydrogen.
[0085] In various aspects, X and Z can independently be an antibiotic, antimicrobial, growth
factor, chemotherapeutic agent, and combinations thereof. A listing of classes and
specific drugs suitable for use in the present invention may be found in
Pharmaceutical Drugs: Syntheses, Patents, Applications by Axel Kleemann and Jurgen
Engel, Thieme Medical Publishing, 1999 and the
Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, Ed. by Budavari
et al., CRC Press, 1996, both of which are incorporated herein by reference. Examples of such pharmaceutically
active agents are provided in the Tables appended hereto. Such pharmaceutically active
agents can be delivered to particular organs, tissues, cells, extracellular matrix
components, and/or intracellular compartments via any suitable method, including the
use of a functional group such as an antibody, antibody fragment, aptamer, and so
on.
[0086] For instance, X can independently include doxorubicin, camptothecin, gemicitabine,
carboplatin, oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin,
methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,
6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate,
melphalan, vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin,
cyclophosphamide, paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel,
maytansanol, epothilone A, combretastatin, pharmaceutically active analogs thereof,
and pharmaceutically acceptable salts thereof.
[0087] These and other pharmaceutically active agents can be covalently conjugated by a
suitable chemical linker through environmentally cleavable bonds. Any of a variety
of methods can be used to associate a linker with a pharmaceutically active agent
including, but not limited to, passive adsorption (
e.g., via electrostatic interactions), multivalent chelation, high affinity non-covalent
binding between members of a specific binding pair, covalent bond formation, etc.
In some embodiments, click chemistry can be used to associate a linker with a particle
(e.g. Diels-Alder reaction, Huigsen 1,3-dipolar cycloaddition, nucleophilic substitution,
carbonyl chemistry, epoxidation, dihydroxylation, etc.). In various aspects, drug
conjugates including a plurality of pharmaceutically active agents, each of which
is covalently bound to a linker, wherein the conjugate releases the pharmaceutically
active agent upon delivery to target cells, are provided.
[0088] Some chemical bonds such as hydrazone, ester and amide bonds are sensitive to acidic
pH values, for example, of the intracellular environment of tumor cells. At acidic
pH, hydrogen ions catalyze the hydrolysis of these bonds which in turn releases the
drug from its conjugate format. Therefore, different pharmaceutically active agents,
such as but not limited to paclitaxel, gemcitabin, doxorubicine, cisplatin, docetaxel,
etc, having -OH, -NH
2, and/or ketonic groups may be covalently linked together with a suitable spacer with
alkyl chains of variable lengths. These spacers may be easily introduced to the drug
conjugates by reacting different acid anhydrides and any organic compounds having
monofunctional or bifunctional or hetero functional groups with the drugs.
[0089] For the pharmaceutically active agents without functional groups such as-OH, -NH
2, or ketonic groups, they may be covalently linked with other pharmaceutically active
agents by creating such functional groups. For example, cisplatin can first be oxidized
to its hydroxyl derivative which then can react with carboxylic acid aldehyde or acid
anhydride to create an aldehydic and carboxylic functional group. This functional
group can be covalently linked with other drugs with -OH and/or -NH
2. Many pharmaceutically active agents can be linked together to form combinatorial
drug conjugates for combination therapy. Those of skill in the art are able to recognize
other conjugation methods which are well known in the art. Such conjugation methods
may be used to link various pharmaceutically active agents, including small molecules,
polypeptides, and polynucleotides, via linkers, including stimuli-sensitive linkers.
[0090] In various aspects, the variable 'n' of the formula (X-Y-Z)
n is an integer greater than or equal to 2. In various aspects, this numeral represents
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50 and even greater numbers of drug-linker and drug-drug conjugates
can be contained in the nanoparticle.
[0091] In another aspect, each individual conjugated drug of the combination comprises a
predetermined molar weight percentage from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, to about 99%, provided that the sum of all individual conjugated
drug molar weight percentages of the combination is 100%. For example, a first drug-linker
conjugate can comprise 70 weight percent (70% w/w) and a second drug-linker conjugate
can comprise 30 weight percent (30% w/w) as contained in the nanoparticle. In another
example, a first drug-drug conjugate can comprise 40 weight percent (40% w/w) and
a second drug-linker conjugate can comprise 60 weight percent (60% w/w) as contained
in the nanoparticle. In yet another example, a first drug-linker conjugate can comprise
10 weight percent (10% w/w), a second drug-linker conjugate can comprise 30 weight
percent (30% w/w), and a third drug-linker conjugate can comprise 60 weight percent
(60% w/w) as contained in the nanoparticle. As another example, a first drug-drug
conjugate can comprise 10 weight percent (10% w/w), a second drug-drug conjugate can
comprise 30 weight percent (30% w/w), and a third drug-drug conjugate can comprise
60 weight percent (60% w/w) as contained in the nanoparticle.
[0092] By using predetermined molar weight percentages, precise ratios among conjugated
drugs in the nanoparticle can be provided. For example, among two-drug conjugate combinations,
ratios including 1:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,
170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,
272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 300, 301, 302, 303, 304,
305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,
322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,
339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,
356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,
390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,
407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,
424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,
458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,
475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,
492, 493, 494, 495, 496, 497, 498, 499, and 1:500 are provided. In another example
having three-drug conjugate combinations ratios of 1:1:1, 1:2:1, 1:3:1, 1:1:2, 1:1:3,
and so forth are provided. Those of skill in the art will recognize that other ratios
can be provided with different numbers of drugs and different molar weight percentages
are utilized.
[0093] In various aspects, Z can independently be an antibiotic, antimicrobial, growth factor,
chemotherapeutic agent, hydrogen, and combinations described above. In addition, Z
can be hydrogen (e.g., a drug-linker conjugate).
[0094] In various aspects, Y is a pH-sensitive linker. For instance, Y can include C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0095] In various aspects, the outer surface of the nanoparticle can include a cationic
or anionic functional group.
[0096] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula I:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W' is phenyl or tert-butyl oxy; and 'R' is hydrogen or alkyl. For instance,
'p' can be 3; 'X' can be chloride; 'W' can be phenyl and 'R' can be hydrogen.
[0097] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula II:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W
1' and 'W
2' are independently selected from phenyl or tert-butyl oxy; and 'R' is hydrogen or
alkyl. For instance, 'p' can be 3; 'X' is chloride; 'W
1' and 'W
2' can be phenyl and 'R' can be hydrogen.
[0098] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula III:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; and 'W' is slected from phenyl or tert-butyl oxy. For instance, 'p' can be 3;
and 'W' can be phenyl.
[0099] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula IV:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy; and 'V
1' and 'V
2' are independently selected from -CH
3 or -CH
2OH. For instance, 'W' can be phenyl; and 'V
1' and 'V
2' can be -CH
2OH.
[0100] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula V:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy. For instance, 'W' can be phenyl.
[0101] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula VI:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can be
phenyl.
[0102] In yet another aspect, the conjugated drug of the combination contained in the nanoparticle
inner sphere has Formula VII:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0103] In various aspects, the nanoparticle can be about 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,
170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,
272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 300, 301, 302, 303, 304,
305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,
322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,
339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,
356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,
390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,
407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,
424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,
458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,
475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,
492, 493, 494, 495, 496, 497, 498, 499, 500, 600, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000, and about 10000 nm in diameter. In various aspects, and particularly depending
on the route of administration in a subject, the nanoparticle can have a diameter
from about 30 nm to about 300 nm. In general, larger nanoparticles are acceptable
when administered locally or topically where the nanoparticle is not required to traverse
a subject vasculature to contact a target cell, tissue or organ. Likewise, smaller
nanoparticles are acceptable when administered systemically in a subject, in particular
nanoparticles from about 30 nm to about 300 nm.
Multi-Drug Conjugates
[0104] In another embodiment, a multi-drug conjugate is provided having the following formula:
X-Y-Z
wherein X and Z are pharmaceutically active agents independently selected from the
group consisting of an antibiotic, antimicrobial, growth factor, and chemotherapeutic
agent; and Y is a stimuli-sensitive linker, wherein the conjugate releases at least
one pharmaceutically active agent upon delivery of the conjugate to a target cell.
Such conjugated drugs are provided above as contained in the nanoparticle of the present
invention.
[0105] In various aspects of the present embodiment, Y is a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof. For instance, Y can be a C
3 straight chain alkyl or a ketone. In various aspects, the pharmaceutically active
agent comprises an anticancer chemotherapy agent. For instance, X and Y can independently
be doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, or pharmaceutically acceptable salts thereof.
[0106] In yet another aspect, the conjugate has Formula I:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W' is phenyl or tert-butyl oxy; and 'R' is hydrogen or alkyl. For instance,
'p' can be 3; 'X' can be chloride; 'W' can be phenyl and 'R' can be hydrogen.
[0107] In another aspect, the conjugate has Formula II:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; 'X' is selected from the group consisting of halogen, sulfate, phosphate, nitrate,
and water; 'W
1' and 'W
2' are independently selected from phenyl or tert-butyl oxy; and 'R' is hydrogen or
alkyl. For instance, 'p' can be 3; 'X' can be chloride; 'W
1' and 'W
2' can be phenyl and 'R' can be hydrogen.
[0108] In another aspect, the conjugate has Formula III:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 1 to
10; and 'W' is slected from phenyl or tert-butyl oxy. For instance, 'p' can be 3;
and 'W' can be phenyl.
[0109] In another aspect, the conjugate has Formula IV:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy; and 'V
1' and 'V
2' are independently selected from -CH
3 or -CH
2OH. For instance, 'W' can be phenyl; and 'V
1' and 'V
2' can be --CH
2OH.
[0110] In another aspect, the conjugate has Formula V:
and pharmaceutically acceptable salts thereof, wherein 'W' is phenyl or tert-butyl
oxy. For instance, 'W' can be phenyl.
[0111] In another aspect, the conjugate has Formula VI:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
[0112] In another aspect, the conjugate has Formula VII:
and pharmaceutically acceptable salts thereof, wherein 'p' is an integer from 5 to
20; and 'W' is phenyl or tert-butyl oxy. For instance, 'p' can be 10; and 'W' can
be phenyl.
Multi-Linked Drug Conjugates
[0113] In yet another embodiment, a multi-drug conjugate is provided comprising a pharmaceutically
active agent covalently bound to a plurality of stimuli-sensitive linkers, wherein
each linker is covalently bound to at least one additional pharmaceutically active
agent, wherein the conjugate releases at least one pharmaceutically active agent upon
delivery to a target cell. Such conjugates can have a conformation similar to a dendrimer,
and can comprise a series of conjugates in a chain.
[0114] In one aspect, the stimuli-sensitive linker can be a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, or combinations thereof. For instance, the linker can be a
C
3 straight chain alkyl. In yet another instance, the linker can comprise a ketone.
[0115] In yet another aspect, the pharmaceutically active agent comprises anticancer chemotherapy
agents. For instance, the pharmaceutically active agent can include doxorubicin, camptothecin,
gemicitabine, carboplatin, oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin,
aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin,
5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,
etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine,
estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte, 4-desacetylvinblastine,
epothilone B, docetaxel, maytansanol, epothilone A, combretastatin, pharmaceutically
active analogs thereof, and pharmaceutically acceptable salts thereof.
Pharmaceutical Preparations and Methods of Administration
[0116] In another embodiment, a pharmaceutical composition is provided comprising the multi-drug
conjugate above, or a pharmaceutically acceptable salt thereof, in a pharmaceutically
acceptable vehicle.
[0117] The identified nanoparticles and multi-drug conjugates (
i.e. compounds) treat, inhibit, control and/or prevent, or at least partially arrest or
partially prevent, diseases that are treatable by known pharmaceutically active agents
and can be administered to a subject at therapeutically effective doses for the inhibition,
prevention, prophylaxis or therapy for such diseases. The compounds of the present
invention comprise a therapeutically effective dosage of a nanoparticle and/or multi-drug
conjugate, a term which includes therapeutically, inhibitory, preventive and prophylactically
effective doses of the compounds of the present invention and is more particularly
defined below. The subjects treated by administration of the compounds is preferably
an animal, including, but not limited to, mammals, reptiles and avians, more preferably
horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.
Therapeutically Effective Dosage
[0118] Toxicity and therapeutic efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals for determining
the LD
50 (the dose lethal to 50% of the population) and the ED
50, (the dose therapeutically effective in 50% of the population). The dose ratio between
toxic and therapeutic effects is the therapeutic index that can be expressed as the
ratio LD
50/ED
50. Compounds that exhibit large therapeutic indices are preferred. While compounds
exhibiting toxic side effects may be used, care should be taken to design a delivery
system that targets such compounds to the site affected by the disease or disorder
in order to minimize potential damage to unaffected cells and reduce side effects.
[0119] The data obtained from the cell culture assays and animal studies can be used in
formulating a range of dosages for use in humans and other mammals. The dosage of
such compounds lies preferably within a range of circulating plasma or other bodily
fluid concentrations that include the ED
50 with little or no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized. For any compound
of the invention, the therapeutically effective dose can be estimated initially from
cell culture assays. A dosage may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC
50 (the concentration of the test compound that achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be used to more accurately
determine useful dosages in humans and other mammals. Compound levels in plasma may
be measured, for example, by high performance liquid chromatography.
[0120] The amount of a compound that may be combined with a pharmaceutically acceptable
carrier to produce a single dosage form will vary depending upon the host treated
and the particular mode of administration. It will be appreciated by those skilled
in the art that the unit content of a compound contained in an individual dose of
each dosage form need not in itself constitute a therapeutically effective amount,
as the necessary therapeutically effective amount could be reached by administration
of a number of individual doses. The selection of dosage depends upon the dosage form
utilized, the condition being treated, and the particular purpose to be achieved according
to the determination of those skilled in the art.
[0121] The dosage regime for treating a disease or condition with the compounds of the invention
is selected in accordance with a variety of factors, including the type, age, weight,
sex, diet and medical condition of the patient, the route of administration, pharmacological
considerations such as activity, efficacy, pharmacokinetic and toxicology profiles
of the particular compound employed, and whether a compound delivery system is utilized.
Thus, the dosage regime actually employed may vary widely from subject to subject.
Formulations and Use
[0122] The compounds of the present invention may be formulated by known methods for administration
to a subject using several routes which include, but are not limited to, parenteral,
oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and ophthalmic routes. The individual compounds may also be
administered in combination with one or more additional compounds of the present invention
and/or together with other pharmaceutically active or inert agents. Such pharmaceutically
active or inert agents may be in fluid or mechanical communication with the compound(s)
or attached to the compound(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophillic
or other physical forces. It is preferred that administration is localized in a subject,
but administration may also be systemic.
[0123] The compounds of the present invention may be formulated by any conventional manner
using one or more pharmaceutically acceptable carriers. Thus, the compounds and their
pharmaceutically acceptable salts and solvates may be specifically formulated for
administration,
e.
g., by inhalation or insufflation (either through the mouth or the nose) or oral, buccal,
parenteral or rectal administration. The compounds may take the form of charged, neutral
and/or other pharmaceutically acceptable salt forms. Examples of pharmaceutically
acceptable carriers include, but are not limited to, those described in
REMINGTON'S PHARMACEUTICAL SCIENCES (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736
(2005), incorporated herein by reference in its entirety.
[0124] The compounds may also take the form of solutions, suspensions, emulsions, tablets,
pills, capsules, powders, and the like. Such formulations will contain a therapeutically
effective amount of the compound, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper administration to the patient.
The formulation should suit the mode of administration.
Parenteral Administration
[0125] The compound may be formulated for parenteral administration by injection,
e.g., by bolus injection or continuous infusion. Formulations for injection may be presented
in unit dosage form in ampoules or in multi-dose containers with an optional preservative
added. The parenteral preparation can be enclosed in ampoules, disposable syringes
or multiple dose vials made of glass, plastic or the like. The formulation may take
such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0126] For example, a parenteral preparation may be a sterile injectable solution or suspension
in a nontoxic parenterally acceptable diluent or solvent (
e.g., as a solution in 1,3-butanediol). Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
In addition, sterile, fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil may be employed including synthetic mono-
or diglycerides. In addition, fatty acids such as oleic acid may be used in the parenteral
preparation.
[0127] Alternatively, the compound may be formulated in powder form for constitution with
a suitable vehicle, such as sterile pyrogen-free water, before use. For example, a
compound suitable for parenteral administration may comprise a sterile isotonic saline
solution containing between 0.1 percent and 90 percent weight per volume of the compound.
By way of example, a solution may contain from about 5 percent to about 20 percent,
more preferably from about 5 percent to about 17 percent, more preferably from about
8 to about 14 percent, and still more preferably about 10 percent of the compound.
The solution or powder preparation may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the injection. Other methods
of parenteral delivery of compounds will be known to the skilled artisan and are within
the scope of the invention.
Oral Administration
[0128] For oral administration, the compound may take the form of tablets or capsules prepared
by conventional means with pharmaceutically acceptable excipients such as binding
agents, fillers, lubricants and disintegrants:
A. Binding Agents
[0129] Binding agents include, but are not limited to, corn starch, potato starch, or other
starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic
acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives
(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium
carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized
starch, hydroxypropyl methyl cellulose,
(e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Suitable
forms of microcrystalline cellulose include, for example, the materials sold as AVICEL-PH-101,
AVICEL-PH-103 and AVICEL-PH-105 (available from FMC Corporation, American Viscose
Division, Avicel Sales, Marcus Hook, Pennsylvania, USA). An exemplary suitable binder
is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold
as AVICEL RC-581 by FMC Corporation.
B. Fillers
[0130] Fillers include, but are not limited to, talc, calcium carbonate (e.g., granules
or powder), lactose, microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
C. Lubricants
[0131] Lubricants include, but are not limited to, calcium stearate, magnesium stearate,
mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol,
other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil
(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil,
and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof.
Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured
by W.R. Grace Co. of Baltimore, Maryland, USA), a coagulated aerosol of synthetic
silica (marketed by Deaussa Co. of Plano, Texas, USA), CAB-O-SIL (a pyrogenic silicon
dioxide product sold by Cabot Co. of Boston, Massachusetts, USA), and mixtures thereof.
D. Disintegrants
[0132] Disintegrants include, but are not limited to, agar-agar, alginic acid, calcium carbonate,
microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium,
sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized
starch, other starches, clays, other algins, other celluloses, gums, and mixtures
thereof.
[0133] The tablets or capsules may optionally be coated by methods well known in the art.
If binders and/or fillers are used with the compounds of the invention, they are typically
formulated as about 50 to about 99 weight percent of the compound. In one aspect,
about 0.5 to about 15 weight percent of disintegrant, and particularly about 1 to
about 5 weight percent of disintegrant, may be used in combination with the compound.
A lubricant may optionally be added, typically in an amount of less than about 1 weight
percent of the compound. Techniques and pharmaceutically acceptable additives for
making solid oral dosage forms are described in
Marshall, SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker and Rhodes, Eds.),
7:359-427 (1979). Other less typical formulations are known in the art.
[0134] Liquid preparations for oral administration may take the form of solutions, syrups
or suspensions. Alternatively, the liquid preparations may be presented as a dry product
for constitution with water or other suitable vehicle before use. Such liquid preparations
may be prepared by conventional means with pharmaceutically acceptable additives such
as suspending agents (
e.
g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying
agents (
e.g., lecithin or acacia); non-aqueous vehicles (
e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and/or
preservatives (
e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also
contain buffer salts, flavoring, coloring, perfuming and sweetening agents as appropriate.
Preparations for oral administration may also be formulated to achieve controlled
release of the compound. Oral formulations preferably contain 10% to 95% compound.
In addition, the compounds of the present invention may be formulated for buccal administration
in the form of tablets or lozenges formulated in a conventional manner. Other methods
of oral delivery of compounds will be known to the skilled artisan and are within
the scope of the invention.
Controlled-Release Administration
[0135] Controlled-release (or sustained-release) preparations may be formulated to extend
the activity of the compound and reduce dosage frequency. Controlled-release preparations
can also be used to effect the time of onset of action or other characteristics, such
as blood levels of the compound, and consequently affect the occurrence of side effects.
[0136] Controlled-release preparations may be designed to initially release an amount of
a compound that produces the desired therapeutic effect, and gradually and continually
release other amounts of the compound to maintain the level of therapeutic effect
over an extended period of time. In order to maintain a near-constant level of a compound
in the body, the compound can be released from the dosage form at a rate that will
replace the amount of compound being metabolized and/or excreted from the body. The
controlled-release of a compound may be stimulated by various inducers,
e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions
or molecules.
[0137] Controlled-release systems may include, for example, an infusion pump which may be
used to administer the compound in a manner similar to that used for delivering insulin
or chemotherapy to specific organs or tumors. Typically, using such a system, the
compound is administered in combination with a biodegradable, biocompatible polymeric
implant that releases the compound over a controlled period of time at a selected
site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic
acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations
thereof. In addition, a controlled release system can be placed in proximity of a
therapeutic target, thus requiring only a fraction of a systemic dosage.
[0138] As an example, an implantable metronomic infusion pump can be used for local delivery
of the nanoparticles and multi-drug conjugates of the present invention.
See, e.g.,
U.S. Patent Nos. 7,799,016,
7,799,012,
7,588,564,
7,575,574, and
7,569,051, each of which is incorporated herein by reference in its entirety. In this example,
a magnetically controlled pump can be implanted into the brain of a patient and deliver
the nanoparticles and multi-drug conjugates at a controlled rate corresponding to
the specific needs of the patient. A flexible double walled pouch that is formed from
two layers of polymer can be alternately expanded and contracting by magnetic solenoid.
When contracted, the nanoparticles and multi-drug conjugates can be pushed out of
the pouch through a plurality of needles. When the pouch is expanded, surrounding
cerebral fluid is drawn into the space between the double walls of the pouch from
which it is drawn through a catheter to an analyzer. Cerebral fluid drawn from the
patient can be analyzed. The operation of the apparatus and hence the treatment can
be remotely controlled based on these measurements and displayed through an external
controller.
[0139] The compounds of the invention may be administered by other controlled-release means
or delivery devices that are well known to those of ordinary skill in the art. These
include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable
membranes, osmotic systems, multilayer coatings, or a combination of any of the above
to provide the desired release profile in varying proportions. Other methods of controlled-release
delivery of compounds will be known to the skilled artisan and are within the scope
of the invention.
Inhalation Administration
[0140] The compound may also be administered directly to the lung by inhalation. For administration
by inhalation, a compound may be conveniently delivered to the lung by a number of
different devices. For example, a Metered Dose Inhaler ("MDI") which utilizes canisters
that contain a suitable low boiling point propellant,
e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon
dioxide or other suitable gas may be used to deliver a compound directly to the lung.
MDI devices are available from a number of suppliers such as 3M Corporation, Aventis,
Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura.
[0141] Alternatively, a Dry Powder Inhaler (DPI) device may be used to administer a compound
to the lung. DPI devices typically use a mechanism such as a burst of gas to create
a cloud of dry powder inside a container, which may then be inhaled by the patient.
DPI devices are also well known in the art and may be purchased from a number of vendors
which include, for example, Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML
Laboratories, Qdose and Vectura. A popular variation is the multiple dose DPI ("MDDPI")
system, which allows for the delivery of more than one therapeutic dose. MDDPI devices
are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough,
SkyePharma and Vectura. For example, capsules and cartridges of gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix of the compound
and a suitable powder base such as lactose or starch for these systems.
[0142] Another type of device that may be used to deliver a compound to the lung is a liquid
spray device supplied, for example, by Aradigm Corporation. Liquid spray systems use
extremely small nozzle holes to aerosolize liquid compound formulations that may then
be directly inhaled into the lung. For example, a nebulizer device may be used to
deliver a compound to the lung. Nebulizers create aerosols from liquid compound formulations
by using, for example, ultrasonic energy to form fine particles that may be readily
inhaled. Examples of nebulizers include devices supplied by Sheffield/Systemic Pulmonary
Delivery Ltd., Aventis and Batelle Pulmonary Therapeutics.
[0143] In another example, an electrohydrodynamic ("EHD") aerosol device may be used to
deliver a compound to the lung. EHD aerosol devices use electrical energy to aerosolize
liquid compound solutions or suspensions. The electrochemical properties of the compound
formulation are important parameters to optimize when delivering this compound to
the lung with an EHD aerosol device. Such optimization is routinely performed by one
of skill in the art. Other methods of intra-pulmonary delivery of compounds will be
known to the skilled artisan and are within the scope of the invention.
[0144] Liquid compound formulations suitable for use with nebulizers and liquid spray devices
and EHD aerosol devices will typically include the compound with a pharmaceutically
acceptable carrier. In one exemplary embodiment, the pharmaceutically acceptable carrier
is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally,
another material may be added to alter the aerosol properties of the solution or suspension
of the compound. For example, this material may be a liquid such as an alcohol, glycol,
polyglycol or a fatty acid. Other methods of formulating liquid compound solutions
or suspensions suitable for use in aerosol devices are known to those of skill in
the art.
Depot Administration
[0145] The compound may also be formulated as a depot preparation. Such long-acting formulations
may be administered by implantation (e.g., subcutaneously or intramuscularly) or by
intramuscular injection. Accordingly, the compounds may be formulated with suitable
polymeric or hydrophobic materials such as an emulsion in an acceptable oil or ion
exchange resins, or as sparingly soluble derivatives such as a sparingly soluble salt.
Other methods of depot delivery of compounds will be known to the skilled artisan
and are within the scope of the invention.
Topical Administration
[0146] For topical application, the compound may be combined with a carrier so that an effective
dosage is delivered, based on the desired activity ranging from an effective dosage,
for example, of 1.0 µM 1.0 mM. In one aspect of the invention, a topical compound
can be applied to the skin. The carrier may be in the form of, for example, and not
by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository,
pad or gelled stick.
[0147] A topical formulation may also consist of a therapeutically effective amount of the
compound in an ophthalmologically acceptable excipient such as buffered saline, mineral
oil, vegetable oils such as corn or arachis oil, petroleum jelly, Miglyol 182, alcohol
solutions, or liposomes or liposome-like products. Any of these compounds may also
include preservatives, antioxidants, antibiotics, immunosuppressants, and other biologically
or pharmaceutically effective agents which do not exert a detrimental effect on the
compound. Other methods of topical delivery of compounds will be known to the skilled
artisan and are within the scope of the invention.
Suppository Administration
[0148] The compound may also be formulated in rectal formulations such as suppositories
or retention enemas containing conventional suppository bases such as cocoa butter
or other glycerides and binders and carriers such as triglycerides, microcrystalline
cellulose, gum tragacanth or gelatin. Suppositories can contain the compound in the
range of 0.5% to 10% by weight. Other methods of suppository delivery of compounds
will be known to the skilled artisan and are within the scope of the invention.
Other Systems of Administration
[0149] Various other delivery systems are known in the art and can be used to administer
the compounds of the invention. Moreover, these and other delivery systems may be
combined and/or modified to optimize the administration of the compounds of the present
invention.
Ratiometric Control of Drug-Linker and Drug-Drug Compositions in a Nanoparticle
[0150] In yet another embodiment, a method is provided for controlling ratios of conjugated
drugs contained in a nanoparticle inner sphere, the method comprising: a) synthesizing
a combination of a first drug independently conjugated to a stimuli-sensitive linker,
and a second drug independently conjugated to a linker having the same composition,
wherein the first drug conjugate and second drug conjugate have a predetermined ratio;
b) adding the combination to an agitated solution comprising a polar lipid; and c)
adding water to the agitated solution, wherein nanoparticles are produced having a
controlled ratio of conjugated drugs contained in the inner sphere. Unlike other methods
that require several additional steps to create nanoparticles, the present self assembly
of the nanoparticles containing combinations of conjugated drugs is highly efficient.
[0151] In one aspect, the first drug and the second drug can independently include an antibiotic,
antimicrobial, antiviral, growth factor, chemotherapeutic agent, and combinations
thereof. For instance, the first drug and the second drug are independently selected
from the group consisting of doxorubicin, camptothecin, gemicitabine, carboplatin,
oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate,
methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0152] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the stimuli-sensitive linker is selected from the group consisting of C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0153] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional drug independently
conjugated to a stimuli-sensitive linker having the same composition.
[0154] In yet another embodiment, a method is provided for controlling ratios of conjugated
drugs contained in a nanoparticle inner sphere, the method comprising: a) synthesizing
a combination of (i) a first drug and a second drug conjugated by a first stimuli-sensitive
linker, and (ii) a first drug and a second drug conjugated by a second stimuli-sensitive
linker, wherein the first drug conjugate and second drug conjugate have a predetermined
ratio; b) adding the combination to an agitated solution comprising a polar lipid;
and c) adding water to the agitated solution, wherein nanoparticles are produced having
a controlled ratio of conjugated drugs contained in the inner sphere.
[0155] In one aspect, the first drug and the second drug are independently selected from
the group consisting of an antibiotic, antimicrobial, antiviral, growth factor, chemotherapeutic
agent, and combinations thereof. For instance, the first drug and the second drug
can independently include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin,
epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0156] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the first stimuli-sensitive linker and the second stimuli-sensitive linker can independently
include C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0157] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional conjugate of
a first drug and a second drug conjugated by a stimuli-sensitive linker other than
those present in the combination.
Methods of Synthesizing Drug-Linker and Drug-Drug Conjugate Containing Nanoparticles
[0158] In another embodiment, a method is provided for producing a combination of conjugated
drugs having a predetermined ratio in a nanoparticle, said nanoparticle comprising
an inner sphere, the method comprising: a) adding to an agitated solution comprising
a polar lipid a combination of a first drug independently conjugated to a stimuli-sensitive
linker, and a second drug independently conjugated to a linker having the same composition,
wherein the first drug conjugate and the second drug conjugate have a predetermined
ratio; and b) adding water to the agitated solution, wherein nanoparticles are produced
containing in the inner sphere the conjugated drugs having a predetermined ratio.
In various aspects, the method can further comprise: c) isolating nanoparticles having
a diameter less than about 300 nm.
[0159] In various aspects, the first drug and the second drug are independently selected
from the group consisting of an antibiotic, antimicrobial, growth factor, chemotherapeutic
agent, and combinations thereof. For instance, the first drug and the second drug
can independently include doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin,
epirubicin, idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine,
cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, melphalan,
vinblastine, vincristine, leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide,
paclitaxel, leurositte, 4-desacetylvinblastine, epothilone B, docetaxel, maytansanol,
epothilone A, combretastatin, pharmaceutically active analogs thereof, and pharmaceutically
acceptable salts thereof.
[0160] In yet another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For
instance, the stimuli-sensitive linker can be C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, or combinations thereof.
[0161] In yet another aspect, the combination of conjugated drugs having a predetermined
ratio further comprise a third drug independently conjugated to a stimuli-sensitive
linker having the same composition. In various aspects, the solution comprising a
polar lipid further comprises a functionlalized polar lipid.
[0162] In yet another embodiment, a method is provided for producing a combination of conjugated
drugs having a predetermined ratio in a nanoparticle, said nanoparticle comprising
an inner sphere, the method comprising: a) adding to an agitated solution comprising
a polar lipid a combination of (i) a first drug and second drug conjugated by a first
stimuli-sensitive linker, and (ii) a first drug and a second drug conjugated by a
second stimuli-sensitive linker, wherein the first drug conjugate and second drug
conjugate have a predetermined ratio; and b) adding water to the agitated solution,
wherein nanoparticles are produced containing in the inner sphere the conjugated drugs
having a predetermined ratio. In various aspects, the method can further comprise:
c) isolating nanoparticles having a diameter less than about 300 nm.
[0163] In one aspect, the first drug and the second drug can independently include an antibiotic,
antimicrobial, growth factor, chemotherapeutic agent, and combinations thereof. For
instance, the first drug and the second drug are independently selected from the group
consisting of doxorubicin, camptothecin, gemicitabine, carboplatin, oxaliplatin, epirubicin,
idarubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate,
mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,
podophyllotoxin, etoposide, etoposide phosphate, melphalan, vinblastine, vincristine,
leurosidine, vindesine, estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte,
4-desacetylvinblastine, epothilone B, docetaxel, maytansanol, epothilone A, combretastatin,
pharmaceutically active analogs thereof, and pharmaceutically acceptable salts thereof.
[0164] In another aspect, the stimuli-sensitive linker is a pH-sensitive linker. For instance,
the first stimuli-sensitive linker and the second stimuli-sensitive linker can independently
be C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
[0165] In various aspects of the present embodiment, the combination of conjugated drugs
having a predetermined ratio further comprises at least one additional conjugate of
a first drug and a second drug conjugated by a stimuli-sensitive linker other than
those present in the combination. In various aspects, the solution comprising a polar
lipid further comprises a functionlalized polar lipid. An example of a polar lipid
is a phospholipid as defined herein.
Methods of Treating Diseases and Conditions in a Subject
[0166] The pharmaceutically active agents used in the present invention are known to provide
a certain response when administered to subjects. One of skill in the art will readily
be able to choose particular pharmaceutically active agents to use with the nanoparticles
and multi-drug conjugates to treat certain diseases or conditions, including those
listed in the appended tables. In addition, the literature is replete with examples
of administering pharmaceutically active agents to subjects, especially those regulated
by the government.
[0167] Therefore, a method is provided for treating a disease or condition, the method comprising
administering a therapeutically effective amount of the nanoparticle above to a subject
in need thereof. In one aspect, the disease is a proliferative disease including lymphoma,
renal cell carcinoma, prostate cancer, lung cancer, pancreatic cancer, melanoma, colorectal
cancer, ovarian cancer, breast cancer, glioblastoma multiforme and leptomeningeal
carcinomatosis. In another aspect, the disease is a heart disease including Atherosclerosis,
Ischemic heart disease, Rheumatic heart disease, Hypertensive heart disease, Infective
endocarditis, Coronary heart disease, and Constrictive pericarditis. In another aspect,
the disease is an ocular disease selected from the group consisting of macular edema,
retinal ischemia, macular degeneration, uveitis, blepharitis, keratitis, rubeosis
iritis, iridocyclitis, conjunctivitis, and vasculitis. In another aspect, the disease
is a lung disease including asthma, Chronic Bronchitis, Cystic Fibrosis, Emphysema,
Pneumonia, lung cancer, Primary Pulmonary Hypertension, Pulmonary Arterial Hypertension,
and Tuberculosis. In yet another aspect, the disease includes bacterial infection,
viral infection, fungal infection, and parasitic infection.
[0168] In various aspects of the present embodiment, the nanoparticle is administered systemically.
In another aspect, the nanoparticle is administered locally. In yet another aspect,
the local administration is via implantable metronomic infusion pump.
[0169] In yet another embodiment, a method is provided for treating a disease or condition,
the method comprising administering a therapeutically effective amount of the multi-drug
conjugate above to a subject in need thereof. In one aspect, the disease is a proliferative
disease including lymphoma, renal cell carcinoma, prostate cancer, lung cancer, pancreatic
cancer, melanoma, colorectal cancer, ovarian cancer, breast cancer, glioblastoma multiforme
and leptomeningeal carcinomatosis. In one aspect, the disease is a heart disease including
Atherosclerosis, Ischemic heart disease, Rheumatic heart disease, Hypertensive heart
disease, Infective endocarditis, Coronary heart disease, and Constrictive pericarditis.
In one aspect, the disease is an ocular disease including macular edema, retinal ischemia,
macular degeneration, uveitis, blepharitis, keratitis, rubeosis iritis, iridocyclitis,
conjunctivitis, and vasculitis. In one aspect, the disease is a lung disease including
asthma, Chronic Bronchitis, Cystic Fibrosis, Emphysema, Pneumonia, lung cancer, Primary
Pulmonary Hypertension, Pulmonary Arterial Hypertension, and Tuberculosis. In yet
another aspect, the disease is selected from the group consisting of bacterial infection,
viral infection, fungal infection, and parasitic infection.
[0170] In various aspects of the present embodiment, the multi-drug conjugate is administered
systemically. In another aspect, the multi-drug conjugate is administered locally.
In yet another aspect, the local administration is via implantable metronomic infusion
pump.
Methods of Sequentially Delivering a Pharmaceutically Active Drug to a Target
[0171] In yet another embodiment, a method is provided for sequentially delivering a drug
conjugate to a target cell. Preferably, a combination of drug-drug conjugates having
individual linkers of varying sensitivities is administered in an environment whereby
one individual linker is triggered first, followed by another individual linker triggered
at another condition. Therefore, the method comprises administering a nanoparticle
above to the target cell and triggering multi-drug conjugate release. In various aspects
of the present embodiment, the nanoparticle is administered systemically. In another
aspect, the nanoparticle is administered locally. In yet another aspect, the local
administration is via implantable metronomic infusion pump.
Methods of Nanoencapsulation with High Loading Efficiency
[0172] In yet another embodiment, a method is provided for nanoencapsulation of a plurality
of drugs comprising: separately linking each of the plurality of drugs with a corresponding
polymer backbone with nearly 100% loading efficiency by forming the corresponding
polymer backbone by ring opening polymerization beginning with the corresponding drug,
wherein each of the corresponding polymer backbones has the same or similar physicochemical
properties and has approximately the same chain length; mixing the plurality of linked
drugs and polymers at selectively predetermined ratios at selectively and precisely
controlled drug ratios; and synthesizing the mixed plurality of linked drugs and polymers
into a nanoparticle.
[0173] In various aspects, the plurality of drugs can independently include an antibiotic,
antimicrobial, growth factor, chemotherapeutic agent, and combinations thereof. For
instance, the plurality of drugs can independently include doxorubicin, camptothecin,
gemicitabine, carboplatin, oxaliplatin, epirubicin, idarubicin, carminomycin, daunorubicin,
aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin,
5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,
etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine,
estramustine, cisplatin, cyclophosphamide, paclitaxel, leurositte, 4-desacetylvinblastine,
epothilone B, docetaxel, maytansanol, epothilone A, combretastatin, pharmaceutically
active analogs thereof, and pharmaceutically acceptable salts thereof.
[0174] In various aspects, the polymer backbone is a stimuli-sensitive linker. For instance,
the stimuli-sensitive linker can include a C
1-C
10 straight chain alkyl, C
1-C
10 straight chain O-alkyl, C
1-C
10 straight chain substituted alkyl, C
1-C
10 straight chain substituted O-alkyl, C
4-C
13 branched chain alkyl, C
4-C
13 branched chain O-alkyl, C
2-C
12 straight chain alkenyl, C
2-C
12 straight chain O-alkenyl, C
3-C
12 straight chain substituted alkenyl, C
3-C
12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic
acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl,
aralkyl, heterocyclic, and combinations thereof.
Kits
[0175] In various embodiments, the present invention can also involve kits. Such kits can
include the compounds of the present invention and, in certain embodiments, instructions
for administration. When supplied as a kit, different components of a compound formulation
can be packaged in separate containers and admixed immediately before use. Such packaging
of the components separately can, if desired, be presented in a pack or dispenser
device which may contain one or more unit dosage forms containing the compound. The
pack may, for example, comprise metal or plastic foil such as a blister pack. Such
packaging of the components separately can also, in certain instances, permit long-term
storage without losing activity of the components. In addition, if more than one route
of administration is intended or more than one schedule for administration is intended,
the different components can be packaged separately and not mixed prior to use. In
various embodiments, the different components can be packaged in one combination for
administration together.
[0176] Kits may also include reagents in separate containers such as, for example, sterile
water or saline to be added to a lyophilized active component packaged separately.
For example, sealed glass ampules may contain lyophilized compounds and in a separate
ampule, sterile water, sterile saline or sterile each of which has been packaged under
a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable
material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic,
metal or any other material typically employed to hold reagents. Other examples of
suitable containers include bottles that may be fabricated from similar substances
as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum
or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes,
and the like. Containers may have a sterile access port, such as a bottle having a
stopper that can be pierced by a hypodermic injection needle. Other containers may
have two compartments that are separated by a readily removable membrane that upon
removal permits the components to mix. Removable membranes may be glass, plastic,
rubber, and the like.
[0177] In certain embodiments, kits can be supplied with instructional materials. Instructions
may be printed on paper or other substrate, and/or may be supplied as an electronic-readable
medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape,
audio tape, and the like. Detailed instructions may not be physically associated with
the kit; instead, a user may be directed to an Internet web site specified by the
manufacturer or distributor of the kit, or supplied as electronic mail.
EXAMPLES
[0178] Aspects of the present teachings may be further understood in light of the following
examples, which should not be construed as limiting the scope of the present teachings
in any way.
Example 1- Ratiometric Combinatorial Drug and Nanoparticle Synthesis
Materials.
[0179] L-lactide was purchased from Sigma-Aldrich Co. (Milwaukee, WI), recrystallized three
times in ethylacetate and dried under vacuum. L-lactide crystals were further dried
inside a glove box and sealed into a glass vial under dry argon and then stored at
-20 °C prior to use. 2,6-di-iso-propylaniline (Sigma-Aldrich Co.) and 2,4-pentanedione
(Alfa Aesar Co., Ward Hill, MA) were used as received. All other chemicals and anhydrous
solvents were purchased from Sigma-Aldrich Co. unless otherwise specified. Anhydrous
tetrahydrofuran (THF) and toluene were prepared by distillation under sodium benzophenone
and were kept anhydrous by using molecular sieves. The 2-((2,6-diisopropylphenyl)amino)-4-((2,6-diisopropylphenyl)imino)-2-pentene
(BDI) ligand and the corresponding metal catalysts (BDI)ZnN(SiMe
3)
2 were prepared inside a glove box following a published protocol and stored at -20
°C prior to use (
B. M. Chamberlain, M. Cheng, D. R. Moore, T. M. Ovitt, E. B. Lobkovsky, G. W. Coates,
J Am Chem Soc 2001, 123, 3229-3238). DOX·HCl was purchased from Jinan Wedo Co., Ltd. (Jinan, China) and used as received.
Removal of HCl from DOX·HCl was achieved by neutralizing DOX·HCl solution in water
with triethyleamine, after which the solution color changed from red to purple. The
free base form of DOX was subsequently extracted with dichloromethane. The organic
extract was filtered through anhydrous Na
2SO
4 and dried under vacuum to collect DOX crystals.
(S)-
(+
)-Camptothecine (CPT) was purchased from TCI America and used as received.
Synthesis of 2-((2,6-diisopropylphenyl)amino)-4-((2,6-diisopropylphenyl)imino)-2-pentene
(BDI).
[0180] Ligand BDI was prepared following a previously published protocol with minor modification
(
B. M. Chamberlain, M. Cheng, D. R. Moore, T. M. Ovitt, E. B. Lobkovsky, G. W. Coates,
J Am Chem Soc 2001, 123, 3229-3238). Briefly, 2,6-Di-
n-propylaniline (13.0 mmol) and 2,4-pentanedione (6.5 mmol) in the ratio of 2:1 were
dissolved in absolute ethanol (20 ml). The mixture solution was acidified with concentrated
HCl (0.6 mL) and heated at reflux for 48 h, which resulted in white precipitates.
After being cooled to room temperature, the white precipitates were dissolved with
dichloromethane and saturated aqueous bicarbonate solution. The orange colored solution
was then extracted and washed with brine three times and filtered through anhydrous
Na
2SO
4, followed by being concentrated and precipitated in hexane. The resulting precipitates
were collected by filtration, suspended in diethyl ether (20 mL), and washed with
saturated aqueous bicarbonate followed by brine. The organic layer was then separated
through filtration in the presence of Na
2SO
4 to absorb moisture and then precipitated in hexane as a light brown powder (yield
~ 60%).
1H NMR (JEOL, CDCl
3, 500 MHz):
δ 12.20 (br, 1H, NH), 7.12 (m, 6H, ArH), 4.83 (s, 1H, Hβ), 3.10 (m, 4H, CHMe
2), 1.72 (s, 6H, α-Me), 1.22 (d, 12H, CHMeMe), 1.12 (d, 12H, CHMeMe) ppm. ESI-MS (positive):
m/z = 419.43 [M+H]
+.
Synthesis of (BDI)ZnN(SiMe3)2 catalyst.
[0181] Zinc bis-(trimethylsilyl)amide (463 mg, 1.19 mmol) in toluene (20 mL) was added into
a solution of BDI (500 mg, 1.19 mmol) in toluene (20 mL). The mixture solution was
stirred for 18 h at 80 °C and then the solvent was removed under vacuum to form (BDI)ZnN(SiMe
3)
2 as a light yellow solid, which was recrystallized from toluene at -30 °C to yield
colorless blocks (yield ~ 70%).
1H NMR (JEOL, C
6D
6, 500 MHz):
δ (br, 1H, NH), 6.9-7.13 (m, 6H, ArH), 4.85 (s, 1H,Hβ), 3.25 (m, 4H, CHMe
2), 1.67 (s, 6H, α-Me), 1.1-1.25 (d, 12H+12H=24H, CHMeMe), 0.08-0.1 (18H, s, SiCH
3) ppm.
Ring opening polymerization of l-lactide.
[0182] Following previously published protocols, DOX-PLA and CPT-PLA polymers were synthesized
through ring opening polymerization of l-lactide initiated by alkoxy complex of (BDI)ZnN(SiMe
3)
2 in a glove box under argon environment at room temperature. For the synthesis of
DOX-PLA, (BDI)ZnN(SiMe
3)
2 (6.4 mg, 0.01 mmol) and DOX (5.4 mg, 0.01 mmol) were mixed in 0.5 mL of anhydrous
THF. L-lactide (101.0 mg, 0.7 mmol) dissolved in 2 mL anhydrous THF was added dropwise.
After the l-lactide was completely consumed, the crude product was precipitated in
cold diethyl ether, yielding DOX-PLA conjugates. The CPT-PLA conjugates were synthesized
in the same procedures as the DOX-PLA. These drug-polymer conjugates had a molecular
weight of about 10,000 g/mole determined by gel permeation chromatography.
Synthesis of lipid-coated drug-polymer conjugate nanoparticles.
[0183] Lipid-polymer hybrid nanoparticles with polymeric cores consisting of the synthesized
drug-polymer conjugates were prepared through a nanoprecipitation method (
L. Zhang, J. M. Chan, F. X. Gu, J. W. Rhee, A. Z. Wang, A. F. Radovic-Moreno, F. Alexis,
R. Langer, O. C. Farokhzad, ACS Nano 2008, 2, 1696-1702). In detail, 200 ug of egg PC (Avanti Polar Lipids Inc.) and 260 ug of 1,2- distearoyl-sn-glycero-3-
phosphoethanolamine-N-carboxy(polyethyleneglycol)-2000 (DSPE-PEG-COOH) (Avanti Polar
Lipids Inc.) were dissolved in 4% ethanol and stirred and heated at 68 °C for 3 min.
A total of 500 ug of DOX-PLA and CPT-PLA was dissolved in acetonitrile and added dropwise
to the lipid solution while stirring. The solution was then vortexed for 3 min followed
by the addition of deionized water (1 mL). Then the diluted solution was stirred at
room temperature for 2 h, washed with PBS buffer using an Amicon Ultra centrifugal
filter with a molecular weight cutoff of 100 kDa (Millipore, Billerica, MA), and resuspended
in 1mL of PBS. Nanoparticles with different DOX/CPT drug ratios were prepared by adjusting
the amount of each type of drug-polymer conjugates while keeping the total polymer
weight at 500 ug. The nanoparticle size and surface zeta potential were obtained from
three repeat measurements by dynamic light scattering (DLS) (Malvern Zetasizer, ZEN
3600) with a backscattering angle of 173°. The morphology of the particles was characterized
by scanning electron microscopy (SEM) (Phillips XL30 ESEM). Samples for SEM were prepared
by dropping nanoparticle solution (5 µL) onto a polished silicon wafer. After drying
the droplet at room temperature overnight, the sample was coated with chromium and
then imaged by SEM. The drug loading yield of the synthesized nanoparticles was determined
by UV-spectroscopy (TECAN, infinite M200) using the maximum absorbance at 482 nm for
DOX and 362 nm for CPT. No shift in the absorbance peak was observed between the free
drugs and their polymer conjugates. Standard calibration curves of both DOX and CPT
at various concentrations were obtained to quantify drug concentrations in the nanoparticles.
Cellular colocalization and cytotoxicity studies.
[0184] The MDA-MB-435 cell line was maintained in Dulbecco's modification of Eagle's medium
(DMEM, Mediatech, Inc.) supplemented with 10% fetal calf albumin, penicillin/streptomycin
(GIBCO
®), L-glutamine (GIBCO
®), nonessential amino acids, sodium bicarbonate, and sodium pyruvate (GIBCO
®). The cells were cultured at 37°C and 5% CO
2. For the dual-drug colocalization and cellular internalization study, the cells were
incubated with dual-drug loaded nanoparticles for 4 h, washed with PBS, and fixed
on a chamber slide for fluorescence microscopy imaging. The cytotoxicity of the dual-drug
loaded nanoparticles was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay (Promega, Madison, WI). Briefly, the cells were seeded at 25%
confluency (∼4×10
3 cells/well) in 96-well plates and incubated with different concentrations of drug
loaded nanoparticles for 24 h. The cells were then washed with PBS three times and
incubated in fresh media for an additional 72h. MTT assay was then applied to the
samples to measure the viability of the cells following the manufacturer's instruction.
Results
[0185] In the study, we used (BDI)ZnN(SiMe
3)
2, a metal-amido complex in which BDI refers to 2-((2,6-diisopropylphenyl)amido)-4-((2,6diisopropylphenyl)
-imino)-2-pentene, as a catalyst for the in-situ formation of metal-alkoxide with
the hydroxyl group of DOX and CPT to initiate the living polymerization of l-lactide
and form drug-poly-l-lactide (drug-PLA) conjugates (Figure 2A). The formation of the
drug-polymer conjugates was verified by the
1H-NMR spectroscopy, which exhibits all the characteristic proton resonance peaks corresponding
to the parent drug molecules. The appearance of the aromatic proton resonance at δ
7.5 to 8.0 ppm in DOX-PLA conjugates (Figure 2B, top panel) and δ 7.5 to 8.5 ppm in
CPT-PLA conjugates (Figure 2B, bottom panel) along with the characteristic -CH3 proton
of PLA at δ 1.5 ppm and -CH proton at δ 5.2 ppm confirms the formation of the drug-polymer
conjugates. The desired drug-polymer conjugation products were further validated by
gel permeation chromatography (GPC) which shows the molecular weight as 10,000 Dalton
for both DOX-PLA and CPT-PLA conjugates (Figure 2C). The molecular weight is in accord
with the monomer-to-initiator feed ratio which indicates near 100% conversion of the
monomers to polymers. Since the formation of metal alkoxide complex is quantitative
and the reaction is homogeneous, the reaction proceeded quantitatively such that all
monomers were converted into products. Also the molecular weight of the polymer matches
that from an earlier study conducted by Tong et al. who used (BDI)ZnN(SiMe
3)
2 to catalyze the ring opening polymerization of both DOX and CPT. (
R. Tong, J. Cheng, Bioconjug Chem 2010, 21, 111-121;
R. Tong, J. Cheng, J Am Chem Soc 2009, 131, 4744-4754).
[0186] Upon successful synthesis of the drug-polymer conjugates, we used them to prepare
lipid-polymer hybrid nanoparticles for dual-drug delivery. Using DSPE-PEG and phospholipids
to coat the polymeric nanoparticle core, the resulting lipid-polymer hybrid nanoparticles
are highly stable in water, PBS and serum and have high drug loading yield as the
entire polymeric core consists of the drug-polymer conjugates. Moreover, by simply
adjusting the DOX-PLA:CPT-PLA molar ratio, dual-drug loaded nanoparticles with ratiometric
drug loading of DOX and CPT were prepared. Keeping the total drug-polymer conjugates
weight constant at 1mg, we varied the DOX-PLA:CPT-PLA ratio to tune the ratiometric
drug loading. The resulting drug-loaded nanoparticles exhibit a unimodal size distribution
at ∼100 nm with low PDI values (Figure 3). In addition, the particles possess negative
surface zeta potential, which is consistent with the DSPE-PEG-COOH coating and serves
to prevent the particles from aggregation. The particle size measured by DLS was consistent
with the SEM images of the particles (Figure 3).
[0187] Following the physicochemical characterization of the particles, we next examined
the drug loading efficiency in these drug-polymer conjugate nanoparticle systems.
We prepared various formulations of the nanoparticles with different ratios of drug-polymer
conjugates and found that, in all cases, over 90% of the conjugates were encapsulated
into the nanoparticles (Figure 4). No change in loading efficiency was observed when
DOX-PLA and CPT-PLA conjugates were loaded in combination or separately, presumably
due to the fact that the long and sharply distributed PLA polymer chain gives each
drug molecule a predominant and uniform hydrophobic property. Therefore, they were
completely encapsulated and stabilized by the lipid and the lipid-PEG layers in the
lipid-polymer hybrid nanoparticle system. Furthermore, we varied the DOX-PLA: CPT-PLA
molar ratios from 1:1, to 3:1 and to 1:3, while keeping the total drug-polymer conjugates
mass constant. It was found that the final loading yields of DOX and CPT in the dual-drug
loaded nanoparticles were highly consistent with the initial DOX-PLA: CPT-PLA molar
ratios (supporting information the following table).
TABLE 1 - Characteristic features of the lipid-coated drug-polymer conjugate nanoparticles
DOX-PLA/CPT-PLA molar ratios |
1:0 |
0:1 |
1:1 |
3:1 |
1:3 |
Particle size (nm) |
|
|
100±2 |
|
|
Particle PDI |
|
|
0.17-0.22 |
|
|
Particle zeta potential (mV) |
|
|
-47±2 |
|
|
DOX loading (µM) |
47.8±0.2 |
0 |
24.0+0.1 |
35.8±0.2 |
12.0±0.8 |
CPT loading (µM) |
0 |
48.2±0.1 |
24.4±0.1 |
12.3±0.1 |
36.2±0.2 |
[0188] These results further confirm that this approach enables one to encapsulate different
types of drugs to the same nanoparticles with ratiometric control over drug loading.
[0189] Upon verifying the excellent drug loading efficiency in the present system, we then
examined whether the different drug-polymer conjugates are loaded into the same nanoparticles
as opposed to forming two different particle populations. To this end, we studied
the colocalization of the two drug molecules and their internalization into cells
through fluorescence microscopy. Since DOX is also a highly fluorescent molecule,
the DOX-PLA conjugates can be identified from DOX's characteristic fluorescence wavelength
(excitation/emission = 540 nm/600 nm). To visualize CPT-PLA, we attached a fluorescent
probe, 6-((7-amino-4-mehylcoumarin-3-acetyl) amino) hexanoic acid succinimidyl ester
(excitation/emission = 353 nm/442 nm), to the hydroxyl end of the CPT-PLA. Figure
5A shows the fluorescence microscopy images that exhibit the colocalization of the
DOX-PLA and the CPT-PLA-probe. The colocalization study indicates that no segregation
between the two types of drug-polymer conjugates occurs and each particle contains
both DOX and CPT.
[0190] After having confirmed that the nanoparticles contain a mixture of DOX and CPT, we
next examined the cytotoxicity of these dual-drug loaded nanoparticles in comparison
to the cocktail mixtures of the corresponding single-drug loaded nanoparticles against
MDA-MB-435 breast cancer cells in vitro. The cocktail system was prepared by mixing
DOX-PLA loaded nanoparticles and CPT-PLA loaded nanoparticles at a ratio that is equivalent
to the DOX-PLA:CPT-PLA molar ratio in the dual-drug nanoparticles. Figure 5B shows
the results of IC50 measurements of the dual-drug loaded nanoparticles and cocktail
combination of single-drug loaded nanoparticles. It was found that the dual-drug loaded
nanoparticles consistently showed higher potency as compared to the cocktail systems
for the 3 different drug ratios. In the 3:1, 1:1, and 1:3 DOX-PLA:CPT-PLA combinations,
the dual-drug loaded nanoparticles showed an enhancement in efficacy by 3.5, 2.5,
and 1.1 times, respectively, compared to the cocktail particle mixtures. This enhanced
cytotoxicity of the dual-drug delivery system can be explained, at least partially,
by the fact that dual-drug loaded nanoparticles can deliver more consistent combination
drug payloads when compared to cocktail nanoparticle systems and hence maximize their
combinatorial effect. In the cocktail mixture, variations in the nanoparticle uptake
and the random drug distribution in cells likely compromised the efficacy of the drug
combinations. Figure 5 suggests that the dual-drug loaded nanoparticles enable concurrent
combination drug delivery through particle endocytosis. Once engulfed by the plasma
membrane, nanoparticles are transported by endosomal vesicles before unloading their
drug payloads. This endocytic uptake mechanism is particularly favourable to the drug-polymer
conjugate system used in the present combinatorial drug delivery scheme. The pH drop
associated with endosome maturation subjects the nanoparticles to an acidic environment
and enzymatic digestions, which facilitate the cleavage of the ester linkage between
the drug and the polymers. In addition, the degradation of the polymer PLA releases
lactic acid to further lower the pH surrounding the nanoparticles, thereby further
accelerating the drug release.
[0191] In conclusion, a new and robust approach for combination chemotherapy was presented
by incorporating two different types of drugs with ratiometric control over drug loading
into a single polymeric nanoparticle. By adapting metal alkoxide chemistry, drug conjugated
polymers were synthesized in a quantitative yield with 100% monomer conversion, resulting
in the formation of highly hydrophobic drug-polymer conjugates. These drug-polymer
conjugates were successfully encapsulated into lipid-coated polymeric nanoparticles
with over 90% loading efficiency. Using DOX and CPT as two model chemotherapy drugs,
various ratios of DOX-PLA and CPT-PLA were loaded into the nanoparticles, yielding
particles that are uniform in size, size distribution and surface charge. The cytotoxicity
of these dual-drug carrying nanoparticles was compared with their cocktail their cocktail
mixtures of single-drug loaded nanoparticles and showed superior therapeutic effect.
This strategy can also be exploited for various other chemotherapeutic agents containing
hydroxyl groups as well as different types of combinations for combinatorial treatments
of various diseases. While only two drugs (DOX and CPT) were used to demonstrate the
concept of this combinatorial drug delivery approach, this method can be generalized
to incorporate three or more different types of drugs into the same nanoparticles
with ratiometro control over drug loading.
Example 2 - Synthesis of Multi-Drug Conjugates
Synthesis of PTXL-GEM Conjugates
[0192] Paclitaxel (PTXL) and Gemcitabine hydrochloride (GEM) were purchased from ChemiTek
Company and used without further purification. All other materials including solvents
were purchased from Sigma-Aldrich Company, USA. Single addition luminescence ATP detection
assay for cytotoxicity measurement was purchased from PerkinElmer Inc.
1H NMR spectra were recorded in CDCl
3 using a Varian Mercury 400 MHz spectrometer. Electrospray ionization mass spectrometry
(ESI-MS, Thermo LCQdeca mass spectrometer) and Thermo Fisher Scientific LTQ-XL Orbitrap
mass spectrometer were used to determine the mass and molecular formula of the compounds,
respectively. Reversed phase HPLC purification was performed on an Varian HPLC system
equipped with µ-bonapack C18 column (4.6 mm × 150 mm, Waters Associates, Inc.) using
acetonitrile and water (50/50, v/v) as mobile phase. Thin-layer chromatography (TLC)
measurements were carried out using pre-coated silica gel HLF250 plates (Advenchen
Laboratories, LLC, USA). 4-(
N,N-dimethylamino) pyridinium-4-toluenesulfonate (DPTS) was prepared by mixing saturated
THF solutions of
N,N-dimethylaminopyridine (DMAP) (1 equiv) and
p-toluenesulfonic acid monohydrate (1 equiv) at room temperature. The precipitate was
filtered, washed three times with tetrahydrofuran (THF), and dried under vacuum.
Synthesis of compound 1.
[0193] Paclitaxel (5 mg, 5.8 µmol) and glutaric anhydride (2 mg. 17.5 µmol) were dissolved
in 200 µL dry pyridine. To this solution, DMAP (0.57 µmol) dissolved in 10 µL pyridine
was added and the solution was stirred at room temperature for 3 hrs. The reaction
was monitored by TLC using 9.2/0.8 (v/v) CHCl
3/MeOH as an eluent (product Rf = 0.42). The complete disappearance of the starting
paclitaxel (Rf = 0.54) occurred after 3 hrs of reaction. Then the reaction was quenched
by diluting the solution with dichloromethane (DCM), followed by extracting DMAP and
pyridine with DI water. The remaining dichloromethane solution was concentrated and
precipitated in hexane, resulting in 5.1 mg of the compound 1 as a white powder. The
production yield was about 90%.
1H NMR (CDCl
3, δ ppm) was carried out to characterize the produced compound 1 (Figure 15): 1.14
(s, 3H), 1.25 (s, 3H), 1.69 (s, 3H), 1.9-2.06 (broad, 7H), 2.16-2.27 (br, 4H), 2.2-2.7
(br, 14 H), 3.82 (d, 1H), 4.21 (d,1H), 4.32 (d-1H), 4.48 (t, 1H), 5.0 (d, 1H), 5.5
(d, 1H), 5.69 (d, 1H), 6.0 (d, 1H), 6.3 (br, 2H), 7.09 (d, 1H), 7.3-7.4(m, 7H), 7.5
(m, 3H), 7.6 (m, 1H), 7.74 (d, 2H), 8.13 (d, 2H), 8.6 (s, 1H). The mass of compound
1 was then determined by ESI-MS (positive) m/z 990.29 (M+Na)
+ (Figure 16).
Synthesis of PTXL-GEM conjugate (compound 2)
[0194] Compound 1 (5 mg, 5.2 µmol) was dissolve in 0.5 mL dry DCM containing DTPS (4.6 mg,
15.6 µmol). To the solution, a solution of GEM (1.5 mg, 5.2 µmol) dissolved in 0.5
mL dry N,N-dimethylformamide (DMF) was added and solution was stirred for 15 min.
After 15 min of reaction, DIPC (5 mg, 39 µmol) in 0.1 mL pyridine was added slowly
to the solution and reaction was carried on at room temperature for 24 hrs. The reaction
was monitored by TLC using 9.2:0.8 (v/v) CHCl
3/MeOH as an eluent (product Rf = 0.22). The complete disappearance of the starting
compound 1 (Rf = 0.42) occurred after 24 hrs of reaction. The reaction was then quenched
by diluting the solution with dichloromethane (DCM), followed by extracting DPTS,
DIPC, DMF, and pyridine with DI water. The remaining dichloromethane solution was
concentrated and precipitated in hexane resulting in 6.1 mg of the compound 2 as a
white powder. The production yield was about 86%. The resulting product was purified
by HPLC using acetonitrile/water (50/50, v/v) as an eluent. Then
1H NMR (CDCl
3, δ ppm) was carried out to characterize the produced compound 2 (Figure 10A): 0.91
(s, 1H), 1.14 (s, 3H), 1.22 (s,3H), 1.27(s,3H), 1.62 (s, 7H), 1.67 (s, 3H), 1.9-1.2
(br, 8H), 2.2-2.7 (br, 14H),2.89 (d, 2H), 3.7 (d, 2H), 3.85 (d, 2H), 3.9 (d, 1H),
4.32 (d, 1H), 4.48 (t, 1H), 5.0 (d, 1H), 5.5 (d, 1H), 5.69 (d, 1H), 6.0 (d, 1H), 6.3
(br, 3H) 7.28 (s, 3H), 7.4 (m, 5H), 7.5 (m, 3H), 7.6 (m, 1H), 7.74 (d, 2H), 8.13 (d,
2H), 8.75 (d, 1H), 9.1 (-NH
2, pyrimidine ring). The mass and molecular formula of compound 2 were then determined
by HR-ESI-FT-MS (orbit-trap-MS, positive) m/z 1213.4327 [M+H]
+, 1235.4140 [M+Na]
+. Calcd for C
61H
66F
2N
4O
20 : 1213.4311. Found: 1213.4327 (Figure 10B).
Hydrolysis of PTXL-GEM conjugate (compound 2)
[0195] Hydrolysis study of PTXL-GEM conjugates was performed to confirm that the conjugates
can be hydrolyzed to free PTXL and free GEM and to measure its hydrolysis kinetics
at different pH values. In the study, PTXL-GEM conjugates were incubated in aqueous
solutions with a pH value of 6.0 or 7.4 at 37 °C. At each predefined time interval,
an aliquot of the conjugate solutions was collected and run through HPLC (mobile phase:
acetonitrile/water = 50/50, v/v) to determine the amount of free PTXL, free GEM and
the remaining PTXL-GEM conjugates.
Preperation of drug loaded nanoparticles
[0196] Drug loaded nanoparticles were prepared via nanoprecipitation process. In a typical
experiment, 0.12 mg of lecithin (Alfa® Aesar Co.) and 0.259 mg of 1,2-distearoyl-
sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH,
Avinti® Polar lipids Inc.) was dissolve in 4% ethanol and homogenised to combine the
components and heated at 68 °C for three minutes. To the solution 1 mg of poly(lactic-co-glycolic
acid) (PLGA, M
n = 40 kDa) and calculated amount of drug dissolved in acetonitrile was added dropwise
while heating and stirring. After the addition of PLGA and drug solution, the vial
was vortexes for three minutes followed by the addition of 1 mL of water. The solution
mixture was stirred at room temperature for 2 hrs and washed with Amicon Ultra centrifugal
filter (Millipore, Billerica, MA) with a molecular weight cutoff of 10 kDa and 1 mL
of drug loaded nanoparticles were collected. Bare nanoparticles were prepared similarly
in the absence of drugs. The nanoparticle size and surface ξ-potential were obtained
from three repeat measurements using a dynamic light scattering (Malvern Zetasizer,
ZEN 3600) with backscattering angle of 173°. The morphology and particle size were
further characterized using scanning electron microscopy (SEM). Samples for SEM were
prepared by dropping 5 µL of nanoparticle solutions onto a polished silicon wafer.
After drying the droplet at room temperature overnight, the sample was coated with
chromium and then imaged by SEM. Drug loading yield was determined by using HPLC.
Cellular viability assay
[0197] Cytotoxicity of compound 2 and PTXL-GEM conjugates loaded nanoparticles was assessed
against XPA3 human pancreatic carcinoma cell lines using the ATP assay. First, cells
were seeded (2x10
4) in 96-well plates and incubated for 24 hrs. Next, the medium was replaced with 150
µL of fresh medium and incubated with different concentration of compound 2 dissolved
in DMSO. The final concentration of DMSO in each well was kept constant at 2%. The
plates were then incubated for 72 hrs and measured by ATP reagents following a protocol
provided by the manufacturer. Fresh cell media with 2% DMSO were used as negative
controls. Similar procedures were applied to compare the cytotoxicity of 100 nM of
compound 2 with that of a mixture of free paclitaxel and gemcitabine at the corresponding
drug concentrations at various incubation times including 24 hrs, 48 hrs, and 72 hrs.
Here the use of DMSO is only for solubilizing the free drugs. For the measurement
of the cytotoxicity of PTXL-GEM conjugates loaded nanoparticles, the experiments were
carried out without using DMSO.
Results
[0198] Figure 9 illustrates the synthesis scheme of PTXL-GEM conjugate (compound 2). We
first took advantage of the steric hindrance structural chemistry of PTXL to selectively
convert its 2' hydroxyl group (2'-OH) to a carboxyl moiety (compound 1). PTXL has
three hydroxyl groups, of which two are secondary and one is tertiary. It has been
reported that the tertiary hydroxyl group is highly hindered and unreactive. The secondary
hydroxyl group at 7 position (7-OH) is less reactive than that at 2' position. Typically,
one has to protect the 2'-OH in order to make any modification to the 7-OH group.
Here we used glutaric anhydride (GA) to react with PTXL in the presence of catalytic
amount of
N,N-dimethylaminopyridine (DMAP) for 3 hrs at room temperature
[0199] to selectively modify the 2'-OH resulting in compound 1 as characterized in Figures
14-16. We observed that the reaction had to be limited for 3 hrs with a GA:PTXL molar
ratio of 3:1 for 2'-OH reaction, otherwise (longer reaction time or higher GA:PTXL
ratio) 7-OH reaction occurred. Compound 1 was then reacted with GEM using 1,3-diisopropyl
carbodiimide (DIPC) and 4-(
N,N-dimethylamino) pyridinium-4-toluenesulfonate (DPTS) resulting in the formation of
compound 2. The formation of compound 2 was first confirmed by
1H-NMR spectroscopy with all characteristic peaks and their integration values of PTXL
and GEM, respectively, as indicated in Figure 10A. The 2'-OH reaction was confirmed
by the integration value of 14H for the resonance peaks at δ 2.7-2.2 ppm. These peaks
are corresponding to the methyl protons of acetate groups at C-4 and C-10, the methylene
protons at C-14 position of the PTXL, and the methylene protons of GA linker. The
resonance at δ 2.7-2.2 ppm of unmodified PTXL was integrated as 8H, which increased
to 14H after the conjugation with GA because of the addition of 6H of the methylene
group from GA moiety. In addition, the δ 4.4 ppm of the protons at C-7 position of
PTXL remained intact during the conjugation. This further indicated the PTXL-GA reaction
only occurred at the 2'-OH group as a downfield shifting of C-7 proton would have
appeared if 7-OH reaction had happened. In contrast, a significant downfield shifting
from δ 4.7 to δ 5.5 ppm was observed for the protons at the C-2' position. On the
other hand, the use of GEM in its hydrochloride salt gives exclusive access to its
hydroxyl group, which is thus prone to couple with the carboxyl group in the PTXL-GA
to form an ester bond. In addition, it has been reported that DIPC and DTPS are effective
esterification reagent with high reaction yield. Furthermore, the chemical shift associated
with the -NH
2 protons of the pyrimidine ring at 9.0 ppm were intact after the reaction. This further
confirms that the PTXL-GEM conjugation occurred via ester formation. The resulting
compound 2 was further examined by high resolution mass spectrometry to determine
its mass and molecular formula. As shown in Figure 10B, the results were precisely
consistent with the expected formula of PTXL-GEM conjugates.
[0200] As the ultimate goal of this research is to concurrently deliver dual drugs to the
same cancer cells for combinatorial therapy, it is crucial to ascertain that the linker
bridging the two drugs can be effectively hydrolyzed, thereby releasing individual
drugs to allow them to arrest cancer cells in their independent pathways. The hydrolysis
of PTXL-GEM conjugates was evaluated and confirmed by high performance liquid chromatography
(HPLC) and high resolution mass spectrometry. As shown in Figure 11A, the HPLC chromatogram
clearly showed that after 24 hrs of incubation in water/acetonitrile (75/25, v/v)
solution at pH = 7.4, a portion of the PTXL-GEM conjugates were hydrolyzed to free
PTXL and free GEM with a characteristic HPLC retention time of 6.2 min and 1.8 min,
respectively, which were confirmed by measuring the mass of the compounds collected
at these two retention times (see Figure 17 and 18 for the corresponding mass spectra).
The formation of free PTXL and free GEM upon hydrolysis further evidenced that the
PTXL-GEM conjugation occurred via the coupling of hydroxyl and carboxyl group to form
an ester bond. If the reaction had occurred via amide formation between the -NH
2 of the pyrimidine ring and the carboxyl group, free PTXL and free GEM would not have
been released upon hydrolysis within only 24 hrs. We hypothesize that when these PTXL-GEM
conjugates are delivered to target cells by a drug carrier through endocytosis, the
hydrolysable PTXL-GEM conjugates can be hydrolyzed with a faster rate at the mild
acidic endosomal environment (pH = ~6). To test this hypothesis, we measured the hydrolysis
kinetics of the PTXL-GEM conjugates at pH = 6.0 and 7.4 respectively. As shown in
Figure 11B, the hydrolysis rate was significantly faster at acidic environments (pH
= 6.0) than at neutral solution (pH = 7.4). Near 80% of the drug conjugates were hydrolyzed
to free PTXL and free GEM at pH = 6.0 within the first 10 hrs, while less than 25%
were cleaved at pH = 7.4.
[0201] Next we examined the
in vitro cellular cytotoxicity of free PTXL-GEM conjugates. As both PTXL and GEM are potent
chemotherapy drugs against pancreatic cancer, we chose human pancreatic cancer cell
line XPA3 for this study. Since it has been documented that the 2'-OH group is essential
for high cytotoxicity of PTXL, it is natural to expect that the cytotoxicity profile
of PTXL-GEM conjugates will rely on their hydrolysis process. To test this, we evaluated
the cytotoxicity of the drug conjugates (100 nM concentration) at different hydrolysis
duration, using a mixture of 100 nM free PTXL and 100 nM free GEM as a positive control.
As shown in Figure 11C, large cytotoxicity difference was observed between the drug
conjugates and the free drug mixtures after 24 hrs and 48 hrs incubation, during which
the drug conjugates were partially hydrolyzed. For example, the drug conjugates killed
∼15% of XPA3 cells whereas the drug mixtures killed ∼55% of the cells after 24 hrs
of incubation. However, after 72 hrs of incubation, the cytotoxicity of the PTXL-GEM
conjugates was nearly at the same level as the free PTXL and free GEM mixtures; over
80% of the cells were killed for both systems. This time-dependent cytotoxicity is
consistent with the temporal hydrolysis profile of the PTXL-GEM conjugates at pH =
7.4 measured by HPLC as shown in Figure 11B. It is worth noting that small molecule
drugs such as PTXL, GEM and PTXL-GEM conjugate usually can diffuse across the cell
membranes to the inside of the cells without going through the endocytosis mechanism.
Therefore, the hydrolysis process of PTXL-GEM conjugates follows the pH = 7.4 profile
when the drug conjugates are administered directly without using a drug delivery vehicle.
[0202] After having demonstrated the formation of PTXL-GEM drug conjugates, their spontaneous
hydrolysis to individual drugs, and cytotoxicity against human pancreatic cancer cell
line XPA3, we next loaded the PTXL-GEM conjugates into a recently developed lipid-coated
polymeric nanoparticle to validate the feasibility of using this pre-conjugation approach
to enable nanoparticle dual drug delivery. The PTXL-GEM conjugates were mixed with
poly(lactic-co-glycolic acid) (PLGA) in an acetonitrile solution, which was subsequently
added into an aqueous solution containing lipid and lipid-polyethylene glycol conjugates
to prepare lipid-coated PLGA nanoparticles following a previously published protocol.
L. Zhang, et al.
ACS Nano 2008, 2, 1696. Figure 12A shows a schematic representation of PTXL-GEM conjugates
loaded nanoparticles, which are spherical particles as imaged by scanning electron
microscopy (SEM) (Figure 12B). Dynamic light scattering measurements showed that the
resulting PTXL-GEM conjugates loaded nanoparticles had an unimodel size distribution
with an average hydrodynamic diameter of 70 ± 1.5 nm (Figure 12C), which was consistent
with the findings from the SEM image (Figure 11B). The surface zeta potential of the
drug loaded nanoparticles in water was about -53 ± 2 mV (Figure 12C). We further found
that the size and surface zeta potential of the PTXL-GEM conjugates loaded nanoparticles
were similar to those of the corresponding empty nanoparticles, 70 ± 1 nm and -51
± 2 mV, respectively. This suggests that the encapsulation of PTXL-GEM conjugates
has negligible effect on the formation of the lipid-coated polymeric nanoparticles.
[0203] The encapsulation yield and loading yield of PTXL-GEM conjugates in the nanoparticles
were quantified by HPLC after dissolving the particles in organic solvents to free
all encapsulated drugs. When the initial PTXL-GEM conjugate input was 5 wt%, 10 wt%,
and 15 wt% of the total nanoparticle weight, the drug encapsulation yield was 22.8
± 2.0%, 16.2 ± 0.5%, 10.8 ± 0.7% respectively, which can be converted to the corresponding
final drug loading yield of 1.1 wt%, 1.6 wt%, and 1.6 wt%, respectively (Figure 13A).
Here the drug encapsulation yield is defined as the weight ratio of the encapsulated
drugs to the initial drug input. The drug loading yield is defined as the weight ratio
of the encapsulated drugs to the entire drug-loaded nanoparticles including both excipients
and bioactive drugs. It seemed the maximum PTXL-GEM loading yield was about 1.6 wt%
for the lipid-coated polymeric nanoparticles. This 1.6 wt% drug loading yield can
be converted to roughly 1700 PTXL-GEM drug conjugate molecules per nanoparticle, calculating
from the diameter of the nanoparticle (70 nm), PLGA density (1.2 g/mL) and the molecular
weight of PTXL-GEM conjugate (1212 Da).
[0204] The cytotoxicity of PTXL-GEM conjugates loaded nanoparticles against XPA3 cell lines
was then examined in comparison with free PTXL-GEM conjugates. Figure 13B summarized
the results of IC
50 measurements of PTXL-GEM conjugates loaded nanoparticles and free PTXL-GEM conjugates
for 24 hrs incubation with the cancer cells. It was found that the IC50 value of PTXL-GEM
conjugates was decreased by a factor of 200 for XPA3 cells after loading the drug
conjugates into the lipid-coated polymeric nanoparticles. This enhanced cytotoxicity
of PTXL-GEM conjugates upon nanoparticle encapsulation can be explained, at least
partially, by the fact that nanoparticle drug delivery can suppress cancer drug resistance.
Small molecule chemotherapy drugs that enter cells through either passive diffusion
or membrane translocators are rapidly vacuumed out of the cells before they can take
an effect by transmembrane drug efflux pumps such as P-glycoprotein (P-gp). Drug loaded
nanoparticles, however, can partially bypass the efflux pumps as they are internalized
through endocytosis. Once being engulfed by the plasma membrane, nanoparticles are
transported by endosomal vesicles before unloading their drug payloads. Thus drug
molecules are released farther away from the membrane-bound drug efflux pumps and
therefore are more likely to reach and interact with their targets. The endocytic
uptake mechanism is particularly favourable to the combinatorial drug delivery system
present in this study. The pH drop upon the endosomal maturation into lysosomes will
subject the drug conjugates to more acidic environment and more hydrolase enzymes,
which will facilitate the cleavage of the hydrolysable linkers. Moreover, the degradation
of PLGA polymer will also contribute to lowering the pH value surrounding the nanoparticles
which can accelerate the hydrolysis process of the drug conjugates as well. The enhanced
hydrolysis of the conjugate linkers may also partially answer for the near 200-fold
cytotoxicity increase of PTXL-GEM conjugates after being encapsulated into the nanoparticles.
[0205] While the focus of this article is to report a novel chemical approach to loading
dual chemotherapy drugs into a single nanoparticle for combinatorial drug delivery,
it would be interesting to compare the cytotoxicity of PTXL-GEM conjugates loaded
nanoparticles with that of a cocktail mixture of the same type of nanoparticles containing
either free PTXL or free GEM. However, the vast hydrophobicity (or solubility) difference
between PTXL and GEM makes it practically undoable to load them into the same type
of nanoparticles, such as the lipid-coated polymeric nanoparticles used in this study.
These nanoparticles can encapsulate hydrophobic drugs such as PTXL with high encapsulation
and loading yields but can barely encapsulate hydrophilic drugs such as GEM. In fact,
the inability of loading different drugs to the same type of nanoparticles represents
a generic challenge to many pairs of drugs for combination therapy. The work presented
in this paper may offer a new way to overcome this challenge.
Conclusions
[0206] In conclusion, we have demonstrated the conjugation of PTXL and GEM with a stoichiometric
ratio of 1:1 via a hydrolysable ester linker and subsequently loaded the drug conjugates
into lipid-coated polymeric nanoparticles. The cytotoxicity of the resulting combinatorial
drug conjugates against human cancer cells was comparable to the corresponding free
PTXL and GEM drug mixtures after the conjugates were hydrolyzed. The cytotoxicity
of the drug conjugates was significantly improved after being encapsulated into drug
delivery nanoparticles. This work provides a new method to load dual drugs to the
same drug delivery vehicle in a precisely controllable manner, which holds great promise
to suppress cancer drug resistance. Similar strategy may be generalized to other drug
combinations. Synthesizing combinatorial drug conjugates with a broad range of stoichiometric
ratios is described above.
Synthesis of Ptxl-Pt(IV) drug conjugates loaded nanoparticles
[0207] Paclitaxel and cisplatin were purchased from ChemiTek Industries Co. (SX, China)
and Sigma-Aldrich Company (St. Louis, MO, USA), respectively, and used without further
purification. All other materials including solvents were purchased from Sigma-Aldrich
Company, USA. Single addition luminescence ATP detection assay was purchased from
PerkinElmer Inc. for cytotoxicity measurement.
1H NMR spectra were recorded in CDCl
3 using a Varian Mercury 500 MHz spectrometer. Electrospray ionization mass spectrometry
(ESI-MS, Thermo LCQdeca mass spectrometer) and Thermo Fisher Scientific LTQ-XL Orbitrap
mass spectrometer were used to determine the mass and molecular formula of the compounds.
Reversed phase high performance liquid chromatography (HPLC) purification was performed
on an Varian HPLC system equipped with µ-bonapack C18 column (4.6 mm×150 mm, Waters
Associates, Inc.) using acetonitrile and water (50/50, v/v) as mobile phase.
Synthesis of cis,trans,cis-PtCl2(OCOCH2CH2CH2COOH)2(NH3)2 prodrug.
[0208] cis,trans,cis-PtCl
2(OH)
2(NH
3)
2 was first synthesized following a previously published protocol, (
R. Kuroda, et al. X-ray and NMR studies of trans-dihydroxo-platinum(IV) antitumor
complexes, J Inorg Biochem 22 (1984) 103-17;
M.D. Hall, et al. The cellular distribution and oxidation state of platinum(II) and
platinum(IV) antitumour complexes in cancer cells, J Biol Inorg Chem 8 (2003) 726-32) which was then used to prepare
cis,trans,cis-PtCl
2(OCOCH
2CH
2CH
2 COOH)
2(NH
3)
2. Briefly, an excess of glutaric anhydride was added to an methylene chloride (MC)
solution containing 100 mg (0.3 mmol) of PtCl
2(OH)
2(NH
3)
2 under reflux condition in the presence of catalytic amount of triethylamine (TEA).
After 12 h of reaction, cold water was added to hydrolyze excess glutaric anhydride.
The reaction mixture was kept at 2°C for 16 hrs. The MC was then removed from the
reaction mixture under reduced pressure resulting in a white residue. The residue
was purified by washing with water, ethanol, and ether in that order. The final production
yield was about 45%. The mass and molecular formula of
cis,trans,cis-PtCl
2(OCOCH
2CH
2CH
2COOH)
2(NH
3)
2 were then determined by HR-ESI-FT-MS (orbit-trap-MS, negative) m/z 560.97 [M-H]
-, 596.83 [M+Cl]
+. Calcd for C
10H
20Cl
2N
2O
8Pt : 561.02. Found: 561.97 (see Figure 24).
Synthesis of Ptxl-Pt(IV) conjugate.
[0209] cis,trans,cis-PtCl
2(OH)
2(NH
3)
2 (10 µmol) and Ptxl (6 µmol) were dissolved in 200 µL dry MC.
N, N-dimethylaminopyridine (DMAP, 0.57 µmol) and
N, N-dicyclohexylcarbodiimide (DCC, 50 µmol) dissolved in 100 µL of dry MC were then added
to this solution. The mixture solution was stirred at room temperature for 24 h. The
reaction was monitored by HPLC using 50/50 (v/v) acetonitrile/water as an eluent (product
retention time = 4.5 min). The complete disappearance of the starting paclitaxel (retention
time = 5.5 min) occurred after 24 h of reaction. Solvent was concentrated and the
byproduct dicyclohexylurea (DCU) was removed by filtration. The remaining solvent
was completely removed and the residue was suspended in ethyl acetate and kept at
4 °C, during the process additional DCU precipitates out to form crystals which were
further removed by filtration. The washing process was repeated three times to completely
remove DCU. Finally, Ptxl-Pt(IV) conjugate was precipitated in hexane to obtain yellowish
white powder. The final product was purified by HPLC with a recovery yield of 55%.
1H NMR (CDCl
3,
δ ppm) was carried out to characterize the produced Ptxl-Pt(IV) conjugate: 1.14 (s,
3H), 1.25 (s, 3H), 1.69 (s, 3H), 1.7-2.06 (broad, 9H), 2.16-2.27 (br, 4H), 2.3-2.7
(br, 9H), 2.9 (d, 1H), 3.2-3.6 (br,14H), 4.32 (d, 1H), 4.48 (t, 1H), 5.0 (d, 1H),
5.5 (d, 1H), 5.69 (d, 1H), 6.2-6.3 (br, 2H), 7.09 (d, 1H), 7.3-7.5(m, 10H), 8.13 (d,
2H), 8.6 (-NH), 11.0 (-COOH). The mass and molecular formula of Ptxl-Pt(IV) conjugate
were determined by HR-ESI-FT-MS (orbit-trap-MS, negative) m/z 1395.32 [M-H]
-, Calcd for C
57H
69Cl
2N
3O
21Pt : 1396.34. Found: 1396.32.
Preparation and characterization of Ptxl-Pt(IV) drug conjugates loaded nanoparticles.
[0210] Ptxl-Pt(IV) conjugates were loaded into lipid-coated polymeric nanoparticles through
a nanoprecipitation process. Typically, 0.12 mg of lecithin (Alfa® Aesar Co.) and
0.259 mg of 1,2-distearoyl-
sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH,
Avinti® Polar lipids Inc.) were dissolved in 4% ethanol aqueous solution and heated
at 68 °C for three minutes. Then 1 mg of poly(lactic-co-glycolic acid) (PLGA,
Mn=40 kDa) and calculated amount of Ptxl-Pt(IV) conjugates dissolved in acetonitrile
were added drop-wise into the lipid solution under heating and stirring. After the
addition of PLGA and Ptxl-Pt(IV) conjugate solution, the mixture was vortexed for
3 min followed by the addition of 1 mL of water. The resulting solution was stirred
at room temperature for 2 h and washed with Amicon Ultra centrifugal filter (Millipore,
Billerica, MA) with a molecular weight cutoff of 10 kDa. Finally, 1 mL of Ptxl-Pt(IV)
conjugates loaded nanoparticles were collected. The nanoparticle size was obtained
from three repeat measurements using a dynamic light scattering (Malvern Zetasizer,
ZEN 3600) with backscattering angle of 173°. The morphology and particle size were
further characterized using scanning electron microscopy (SEM). Samples for SEM were
prepared by dropping 5 µL of nanoparticle solutions onto a polished silicon wafer.
After drying the droplet at room temperature overnight, the sample was coated with
chromium and then imaged by SEM. Drug loading yield of the nanoparticles was determined
by using HPLC.
Cellular viability assay.
[0211] Cytotoxicity of free Ptxl-Pt(IV) conjugates and Ptxl-Pt(IV) conjugates loaded nanoparticles
were assessed against A2780 ovarian carcinoma cell lines using the ATP assay. First,
cells were seeded to 10% confluency (∼5x10
3/well) in 96-well plates and incubated for 24 h. Prior to the experiment, the culture
medium was replaced with 150 µL fresh medium and cells were incubated with different
concentration of free Ptxl-Pt(IV) conjugates and Ptxl-Pt(IV) conjugates loaded nanoparticles
for 24 h, followed by washing the cells with PBS to remove excess drugs or nanoparticles.
The cells were then incubated in fresh medium for 72 h and measured by ATP assay following
a protocol provided by the manufacturer. Fresh culture medium was used as a negative
control in this study.
Results
[0212] Figure 19 illustrates the synthesis scheme of Ptxl-Pt(IV) conjugate. We started the
synthesis with the oxidation of cisplatin to form dihydroxy cisplatin, a Pt(IV) prodrug,
which was later conjugated to Ptxl via a glutaric acid linker. In order to conjugate
dihydroxy cisplatin with Ptxl, one can choose to first activate dihydroxy cisplatin
with glutaric anhydride, followed by conjugating the resulting organo platinum complex
to Ptxl. Alternatively, the conjugation can be carried out in a reverse order, where
glutaric anhydride is reacted with Ptxl first and then conjugated to dihydroxy cisplatin.
The difference between these two synthetic routes is that the former involves the
conjugation of an organic compound with an organo platinum complex, while the latter
involves a reaction between an organic compound with a dihydroxy platinum complex.
Given the high flexibility to select proper reaction solvent for an organo platinum
complex and Ptxl as compared to a dihydroxy platinum complex and Ptxl, we chose the
first route to synthesize Ptxl-Pt(IV) hydrophobic-hydrophilic drug conjugates as shown
in Figure 19.
[0213] As discussed in previous paragraph we converted Pt(IV) complex to Carboxyl functionalized
organo Pt complex by reacting with GA (Supporting information Figure 24). Taking an
advantage of the steric hindrance structural chemistry of Ptxl, we selectively reacted
its 2' hydroxyl group (2'-OH) to a carboxyl moiety of Pt(IV) organo Pt complex. Among
three -OH groups in Ptxl, it has been reported that the tertiary hydroxyl group is
highly hindered and unreactive. The secondary hydroxyl group at 7 position (7-OH)
is less reactive than that at 2' position. Typically, one has to protect the 2'-OH
in order to make any modification to the 7-OH group.
[0214] The formation of Ptxl-Pt(IV) hydrophobic and hydrophilic conjugate was first confirmed
by
1H-NMR spectroscopy with all characteristic peaks and their integration values of Ptxl
and Pt(IV), respectively, as indicated in Figure 20A. The reaction at 2'-OH was confirmed
due to the significant downfield shifting of the protons at the C-2' from δ 4.7 to
δ 5.7 ppm. This shifting further confirms esterification between Ptxl and GA functionalized
Pt(IV) thereby confirming the conjugation of Ptxl and Pt(IV) with hydrolysable linker.
However, the protons at C-7 position of Ptxl remained intact at δ 4.4 ppm during the
conjugation, this further indicated the Ptxl-Pt(IV) reaction only occurred at the
2'-OH group as a downfield shifting of C-7 proton would have appeared if 7-OH reaction
had happened. The resulting compound 2 was further examined by high resolution mass
spectroscopy to determine its mass and molecular formula. As shown in Figure 20B,
the results were consistent with the expected formula of Ptxl-Pt(IV) conjugate. However,
one might expect two molecules of Ptxl to attach to GA functionalized Pt(IV) due to
presence of two -COOH group. Such conjugation was not observed likely because one
of the GA moiety at the axial position became sterically hindered after one Ptxl was
attached. The 1:1 conjugation was further confirmed by the corresponding molecular
formula for Ptxl-Pt(IV) and the appearance of -COOH proton resonance at δ 11.0 ppm.
[0215] Upon completion of the conjugate synthesis and characterization, the Ptxl-Pt(IV)
compound was subsequently loaded into a recently developed lipid-coated polymeric
nanoparticleas demonstrated in Figure 21A to confirm whether co-encapsulation of hydrophobic
and hydrophilic drugs can be accomplished using this pre-conjugation approach. Based
on a previously published protocol (
L. Zhang, et al. Self-assembled lipid--polymer hybrid nanoparticles: a robust drug
delivery platform, ACS Nano 2 (2008) 1696-702), the Ptxl-Pt(IV) conjugates were mixed with poly(lactic-co-glycolic acid) (PLGA,
Mn=40,000) in an acetonitrile solution, which was then added drop-wise in aqueous
solution containing lipid and lipid-polyethylene glycol conjugates to prepare lipid-coated
PLGA nanoparticles (Figure 21A). To quantify the loading yield of Ptxl-Pt(IV) conjugates,
the nanoparticles were dissolved in organic solvents to free all encapsulated drugs.
The solution was then analyzed by high performance liquid chromatography (HPLC). An
initial Ptxl-Pt(IV) conjugate input of 10 wt% of the total polymeric nanoparticle
weight yielded a final loading of 1.86% (wt/wt), or 18.6µg per 1mg of polymer (Figure
24), which is comparable with published data on nanoparticle drug loading (
J.M. Chan, et al. PLGA-lecithin-PEG core-shell nanoparticles for controlled drug delivery,
Biomaterials 30 (2009) 1627-34). Figure 21A shows a schematic representation of Ptxl-Pt(IV) conjugate loaded nanoparticles,
which are spherical particles with unimodal size distribution with an average hydrodynamic
diameter of 70 nm and a PDI of 0.21 as shown by dynamic light scattering (DLS) measurements
(Figure 21B). SEM images further showed that the resulting Ptxl-Pt(IV) conjugates
loaded nanoparticles had an unimodal size distribution with an average diameter of
70 nm (Figure 21C), which was consistent with the findings from DLS (Figure 21B).
[0216] After having demonstrated the loading of Ptxl-Pt(IV) conjugate, we next evaluated
the in-vitro cellular cytotoxicity of Ptxl-Pt(IV) against A2780 human ovarian cancer
cells as shown in Figure 22. The cells were incubated with free Pxtl-Pt(IV) conjugates
and Pxtl-Pt(IV) conjugates in nanoparticles at different concentrations for 4hrs followed
by PBS washing and incubation in fresh media for 72hrs before ATP cell viability assay
(Figure 22A). It was observed that the Ptxl-Pt(IV) showed less toxicity as compared
to that of Ptxl-Pt(IV) loaded nanoparticles. This reduced toxicity could be attributed
to several factors. Firstly, the conjugation of a hydrophobic Ptxl and a hydrophilic
Cisplatin gives rise to a large amphiphilic molecule that is structurally similar
to phospholipids. The amphiphilic conjugate is more likely to be anchored in the lipid
bilayer, resulting in less efficient drug delivery. Secondly, the cytoplasmic pH of
cancer cells, which is approximately 6.8 to 7.1, cannot efficiently break the ester
bond that connects the two drug molecules. In the conjugate form Ptxl and Pt(IV) cannot
freely interact with their molecular targets. Therefore a slow hydrolysis rate will
significantly compromise the conjugate's potency.
[0217] Cytotoxicity of the Pxtl-Pt(IV) conjugate-loaded nanoparticles provides evidence
that both membrane diffusion and conjugate hydrolysis issues can be overcome by nanoparticle
delivery. As shown in Figure 22A, large toxicity difference was observed between the
free Ptxl-Pt(IV) and Ptxl-Pt(IV) loaded NPs system. Such difference can be easily
observed from the microscopic images of the cells after the treatment with free Ptxl-Pt(IV)
and Ptxl-Pt(IV) loaded NPs as shown in Figure 22B and C, respectively. The number
of viable cells were significantly reduced after the treatment with Ptxl-Pt(IV) loaded
NPs, Figure 2C. It has been well studied that nanoparticles below 100nm in size are
taken up by cells through endocytic uptake. Upon contact with the nanoparticles the
cell membranes fold inward and engulf the particles in endocytic vesicles. This process
allows the drug conjugates to efficiently enter the cytoplasm without relying on passive
diffusion through the lipid bilayers, which is highly unfavorable to large amphiphilic
molecules. Another benefit of the endocytic uptake mechanism is that the endo-lysomal
environments provides a more acidic medium which can accelerate the hydrolysis of
the ester linker in the Pxtl-Cisplatin conjugate. As endosomes matures into lysosomes,
their pH can drop to ∼5.5. The excess protons speed up the drug release that unblocks
the functional 2'-OH of the Ptxl and relieves the Pt(IV) which reduced to Cisplatin
in intracellular environment. In addition, the degradation of the PLGA polymers into
lactic acid will further lower the pH value surrounding the nanoparticles, resulting
in even faster drug release. The enhanced toxicity in the nanoparticle formulation
of the Pxtl-Pt(IV) has significant implications as it addresses common issues in drug
conjugates. Additionally, the strategy adds applicability to the fast-growing nanoparticle
platforms and could potentially address the side effects associated with premature
drug release in the circulation as the drug conjugates are much less potent without
the vehicle.
Conclusions
[0218] In conclusion, we have demonstrated the conjugation of hydrophobic Ptxl and hydrophilic
cisplatin with a hydrolysable ester linker and subsequently encapsulated the compound
into a lipid-coated polymeric nanoparticle. The cytotoxicity of the resulting Ptxl-Pt(IV)
conjugates against ovarian cancer cells was compared to the corresponding free Ptxl
and cisplatin drug mixtures after the conjugates were hydrolyzed. The efficacy of
Ptxl-Pt(IV) was significantly improved after being encapsulated into drug delivery
nanoparticles. This work provides a new approach to load hydrophobic and hydrophilic
drug to the same drug delivery vehicle without adding complexity to the nanoparticle
structure. We demonstrate that prodrug conjugates and nanoparticulate systems can
complement each other as an excellent combinatorial drug delivery platform.
TABLE 2 - Exemplary Cancers and Tumors
|
adenocarcinoma, pancreatic |
|
adenocarcinoma, papillary, bladder |
ackerman tumor |
adenocarcinoma, pleomorphic |
adenocarcinoid, malignant, appendiceal |
adenocarcinoma, polymorphous low-grade |
adenocarcinoma variant, gastric cancer |
adenocarcinoma, proximal jejunum |
adenocarcinoma, alpha-fetoprotein-producing, esophageal |
adenocarcinoma, rete testis |
adenocarcinoma, apocrine |
adenocarcinoma, small bowel |
adenocarcinoma, appendiceal |
adenocarcinoma, thymus |
adenocarcinoma, bartholin gland |
adenocarcinoma, unknown primary site |
adenocarcinoma, bladder |
adenocarcinoma, urachal |
adenocarcinoma, clear cell |
adenocarcinoma, urethral |
adenocarcinoma, colloid |
adenocarcinoma, vaginal |
adenocarcinoma, ductal type |
adenomyoepithelioma, malignant, breast |
adenocarcinoma, eccrine |
adenosarcoma, Müllerian |
adenocarcinoma, endometrioid primary, in colorectal endometriosis |
adrenogenital syndrome /testicular tumor |
|
ameloblastoma, desmoplastic |
adenocarcinoma, esophagus |
ameloblastoma, malignant |
adenocarcinoma, fallopian tube |
amyloid |
adenocarcinoma, fetal pulmonary |
angioblastoma, giant cell |
adenocarcinoma, gall bladder |
angioendothelioma, malignant, endovascular papillary |
adenocarcinoma, hepatoid |
angioendotheliomatosis, malignant |
adenocarcinoma, in situ, cervix |
angiomyxoma, malignant, aggressive, scrotum |
adenocarcinoma, intra-extrahepatic, bile ducts |
angiomyxoma, malignant, aggressive, scrotum |
adenocarcinoma, lacrimal gland |
angiosarcoma |
adenocarcinoma, large bowel |
angiosarcoma, cardiac |
adenocarcinoma, low-grade, extraosseous endolymphatic sac |
angiosarcoma, pulmonary artery |
adenocarcinoma, mucinous |
angiosarcoma, Wilson-Jones |
adenocarcinoma, mucinous, prostate |
askin tumor |
adenocarcinoma, mucinous, stomach |
astroblastoma |
adenocarcinoma, oncocytic |
astrocytic neoplasm |
astrocytoma, anaplastic |
carcinoma, adenosquamous |
astrocytoma, gemistocytic |
carcinoma, adenosquamous, liver |
astrocytoma, pilocytic |
carcinoma, adenosquamous, pancreatic |
astrocytoma, thalamic glioma |
carcinoma, adrenocortical |
blastoma, pleuropulmonary (PPB) |
carcinoma, ameloblastic |
blastoma, pulmonary |
carcinoma, anal |
borderline tumor, malignant, ovary |
carcinoma, anaplastic |
Buschke-Lowenstein tumor giant condyloma |
carcinoma, anaplastic, thymic |
calcifying epithelial odontogenic tumor (CEOT) |
carcinoma, anaplastic, thyroid |
carcinamitosis, peritoneal |
carcinoma, apocrine |
carcinoid, malignant |
carcinoma, basal cell, perianal |
carcinoid, malignant, atypical |
carcinoma, basal cell, vulva |
carcinoid, malignant, bronchopulmonary, atypical |
carcinoma, basaloid squamous cell, esophageal |
carcinoid, malignant, bronchopulmonary, typical |
carcinoma, basaloid squamous cell, NOS |
carcinoid, malignant, colorectal |
carcinoma, basaloid, lung |
carcinoid, malignant, gastric |
carcinoma, bile duct |
carcinoid, malignant, gastrointestinal, appendix |
carcinoma, biliary tract |
carcinoid, malignant, goblet cell |
carcinoma, bronchioalveolar (BAC) |
carcinoid, malignant, lung |
carcinoma, bronchogenic small cell undifferentiated |
carcinoid, malignant, pulmonary |
carcinoma, choroid plexus |
carcinoid, malignant, rectal |
carcinoma, ciliated cell |
carcinoid, malignant, renal |
carcinoma, clear cell, bladder |
carcinoid, malignant, small bowel |
carcinoma, clear cell, eccrine |
carcinoid, malignant, thymic |
carcinoma, clear cell, odontogenic |
carcinoma, acinar cell (ACC) |
carcinoma, clear cell, thymic |
carcinoma, acinic cell |
carcinoma, collecting duct (CDC) |
carcinoma, adenoid basal, uterine cervix |
carcinoma, collecting duct, kidney |
carcinoma, adenoid cystic (AdCC) |
carcinoma, cribriform |
carcinoma, adenoid cystic, breast (ACCB) |
carcinoma, cribriform, breast |
carcinoma, adenoid cystic, breast, metastatic (ACC-M) |
carcinoma, cystic |
carcinoma, duodenal |
carcinoma, papillary, breast |
carcinoma, epithelial-myoepithelial (EMC) |
carcinoma, parathyroid |
carcinoma, gall bladder |
carcinoma, parietal cell |
carcinoma, giant cell |
carcinoma, penile |
carcinoma, hepatocellular |
carcinoma, pilomatrix |
carcinoma, Hurthle cell |
carcinoma, pituitary |
carcinoma, Hurthle cell, thyroid |
carcinoma, plasmacytoid urothelial, bladder |
carcinoma, insular |
carcinoma, poorly differentiated, neuroendocrine (PDNEC) |
carcinoma, insular, thyroid |
carcinoma, primary intraosseous |
carcinoma, islet cell |
carcinoma, primary peritoneal, extra-ovarian (EOPPC) |
carcinoma, large cell, neuroendocrine (LCNEC) |
carcinoma, renal cell (RCC), poorly differentiated |
carcinoma, lymphoepithelioma-like, thymic |
carcinoma, renal cell (RCC), chromophobic (ChC) |
carcinoma, male breast |
carcinoma, renal cell (RCC), clear cell (CCC) |
carcinoma, medullary thyroid |
carcinoma, renal cell (RCC), collecting duct (CDC) |
carcinoma, meibomian |
carcinoma, renal cell (RCC), papillary (PC) |
carcinoma, merkel cell (MCC) |
carcinoma, renal cell (RCC), sarcomatoid |
carcinoma, metaplastic, breast |
carcinoma, sarcomatoid, colon |
carcinoma, microcystic adnexal |
carcinoma, sarcomatoid, thymic |
carcinoma, mixed acinar, endocrine |
carcinoma, sebaceous |
carcinoma, moderately differentiated, neuroendocrine |
carcinoma, serous ovarian, papillary(PsOC) |
carcinoma, mucinous, bronchioloalveolar, lung |
carcinoma, signet-ring cell |
carcinoma, mucinous, eccrine |
carcinoma, small cell |
carcinoma, mucoepidermoid |
carcinoma, small cell undifferentiated, prostate |
carcinoma, mucoepidermoid, bronchus |
carcinoma, small cell undifferentiated, prostrate (SCUUP) |
carcinoma, nasopharyngeal/caucasians (NPC) |
carcinoma, small cell, anorectal neuroendocrine |
carcinoma, neuroendocrine |
carcinoma, small cell, colorectal |
carcinoma, neuroendocrine, lung |
carcinoma, small cell, esophageal |
carcinoma, non-small cell w/neuroendocrine features, lung |
carcinoma, small cell, extrapulmonary |
carcinoma, odontogenic |
carcinoma, small cell, gastrointestinal tract |
carcinoma, papillary |
carcinoma, small cell, neuroendocrine (oat cell) (SCNC) |
carcinoma, small cell, pancreatic |
carcinoma, tubal |
carcinoma, small cell, renal |
carcinoma, tubular, breast |
carcinoma, small cell, stomach |
carcinoma, undifferentiated, nasopharyngeal type (UCNT) |
carcinoma, small cell, thymic |
carcinoma, undifferentiated, primary sinonasal nasopharyngea |
carcinoma, small intestine |
carcinoma, undifferentiated, sinonasal (SNUC) |
carcinoma, squamous cell, adnexal ductal cyst |
carcinoma, undifferentiated, thymic |
carcinoma, squamous cell, atypical |
carcinoma, undifferentiated, w/lymphoid stroma |
carcinoma, squamous cell, breast |
carcinoma, vaginal |
carcinoma, squamous cell, diffuse pagetoid, esophagus |
carcinoma, verrucous |
carcinoma, squamous cell, esophageal |
carcinoma, w/ spindle cell metaplasia, breast |
carcinoma, squamous cell, keratinizing, thymic (KTSC) |
carcinoma, w/metaplasia, osteo-chondroid variant, breast |
carcinoma, squamous cell, laryngeal |
carcinoma, w/sarcomatous metaplasia, breast |
carcinoma, squamous cell, lymphoepithelioma-like |
carcinoma, well differentiated, neuroendocrine (WDNEC) |
carcinoma, squamous cell, nasopharynx |
carcinoma, well differentiated, thymic (WDTC) |
carcinoma, squamous cell, nonkeratinizing |
carcinosarcoma |
carcinoma, squamous cell, oral cavity |
carcinosarcoma, uterine |
carcinoma, squamous cell, ovarian |
cartilage tumor |
carcinoma, squamous cell, stomach |
cartilaginous tumor, larynx |
carcinoma, squamous cell, subungual (SCC) |
chemodectoma, malignant |
carcinoma, squamous cell, thymic |
chloroma |
carcinoma, squamous cell, thyroglossal duct cyst (TGDC) |
cholangio-carcinoma |
carcinoma, squamous cell, thyroid |
cholangitis, primary sclerosing |
carcinoma, squamous cell, urethra |
chondroblastoma |
carcinoma, squamous cell, vagina |
chondroid syringoma, malignant (MCS) |
carcinoma, squamous cell, vulvar |
chondroma, malignant, pulmonary (in Carney's triad) |
carcinoma, terminal duct |
chondrosarcoma |
carcinoma, testicular |
chondrosarcoma, acral synovial |
carcinoma, transitional cell |
chondrosarcoma, classic, primary intradural |
carcinoma, transitional cell, prostate |
chondrosarcoma, clear cell |
carcinoma, trichilemmal |
chondrosarcoma, clear cell, larynx |
chondrosarcoma, dural-based |
dermatofibrosarcoma protuberans, fibrosarcomatous variant |
chondrosarcoma, intracranial |
dermatofibrosarcoma protuberans, NOS |
chondrosarcoma, mesenchymal |
dermatofibrosarcoma protuberans, pigmented |
chondrosarcoma, mysoid, extraskeletal |
desmoplastic, small round cell (DSRCT) |
chordoma |
dysembryoplastic neuroepithelial tumor (DNT) |
chordoma, clivus |
dysgerminoma |
chordoma, familial |
dysgerminoma, ovarian |
chordoma, intracranial cavity |
eccrine poroma, malignant |
chordoma, NOS |
eccrine spiradenoma, malignant |
chordoma, perifericum |
ectomesenchymoma, malignant |
chordoma, sacrum |
emlanoma, malignant, placenta |
chordoma, skull base |
endocrine tumor, pancreatic |
chordoma, vertebrae |
endodermal sinus tumor |
choriocarcinoma |
endometrioid tumor, ovary |
choriocarcinoma, esophagus |
ependymoma |
choriocarcinoma, gastric |
epithelial cancer, ovarian (EOC) |
choriocarcinoma, ovary |
epithelial tumor, appendiceal |
choriocarcinoma, stomach |
epithelial tumor, oral cavity |
choriocarcinoma/male, primary, pulmonary |
epithelioma cuniculatum |
cutaneous malignant tumor |
erythroleukemia |
cylindroma, malignant |
esthesioneuroblastoma |
cylindroma, malignant, apocrine |
fibrosarcoma |
cystadenocarcinoma, acinar cell |
fibrous histiocytoma, malignant |
cystadenocarcinoma, mucinous |
fibrous histiocytoma, malignant (MFH) |
cystadenocarcinoma, pancreatic |
fibrous histiocytoma, malignant, angiomatoid |
cystadenocarcinoma, serous |
fibrous histiocytoma, malignant, intracerebral |
cystic-pseudopapillary tumor/ pancreas |
fibrous histiocytoma, malignant, renal |
cystosarcoma phyllodes, malignant, breast |
fibrous tissue tumor, malignant |
cystosarcoma phylloides |
fibrous tumor, solitary, malignant |
dermatofibrosarcoma protuberans (DFSP) |
fibroxanthoma, atypical |
follicular tumor |
hemangiosarcoma |
ganglioneuroblastoma |
hepatoblastoma |
gastrointestinal autonomic nerve tumor |
hereditary non-polyposis colorectal cancer (HNPCC) |
germ cell tumor |
hidradenoma papilliferum, malignant |
germ cell tumor, intracranial (GCTs) |
histiocytoma |
germ cell tumor, ovarian |
histiocytosis, malignant |
germ cell tumor, testicular (GCTS) |
Hodgkin's disease |
germinoma (seminoma) |
Hodgkin's disease, bladder |
germinoma, pineal |
Hodgkin's disease, blood |
gestational trophoblastic tumor |
Hodgkin's disease, bone |
giant cell tumor, nonendocrine |
Hodgkin's disease, bone marrow |
glioblastoma multiforme, spinal chord |
Hodgkin's disease, breast |
glioblastoma, giant cell |
Hodgkin's disease, cardiovascular system |
glioma |
Hodgkin's disease, central nervous system |
glioma, optic nerve |
Hodgkin's disease, connective tissue disease |
glomangiosarcoma |
Hodgkin's disease, endocrine system |
glomus tumor, malignant |
Hodgkin's disease, gastrointestinal tract |
glucagonoma syndrome |
Hodgkin's disease, genitourinary |
granular cell tumor, malignant |
Hodgkin's disease, head & neck |
granular cell tumor, malignant, larynx |
Hodgkin's disease, kidney |
granulosa cell tumor, ovary |
Hodgkin's disease, lung |
granulosa tumor, stromal cell |
Hodgkin's disease, muscle |
gynandroblastoma |
Hodgkin's disease, neurological system |
hamartoma, mesenchymal, liver (MHL) |
Hodgkin's disease, prostate |
hemangioendothelioma |
Hodgkin's disease, reproductive system |
hemangioendothelioma, epithelioid |
Hodgkin's disease, respiratory system |
hemangioendothelioma, spindle cell |
Hodgkin's disease, skin |
hemangioendothelioma, thyroid |
Hodgkin's disease, testis |
hemangioendotheliomas, epithelioid, pulmonary (PEH) |
Hodgkin's disease, thymus |
hemangiopericytoma (HEPC) |
Hodgkin's disease, thyroid |
hypokalemia & achlorhydria syndrome, well differentiated |
leukemia, acute undifferentiated (AUL) |
inflammatory myofibroblastic tumor (IMT) |
leukemia, adult T-cell |
inflammatory myofibroblastic tumor (IMT), pulmonary |
leukemia, basophilic |
insular papillary cancer, thyroid |
leukemia, central nervous system |
insulinoma, malignant |
leukemia, chronic lymphocytic (CLL) |
islet cell tumor, nonfunctioning |
leukemia, chronic myelogenous (CML) |
islet cell, pancreatic |
leukemia, cutis |
Krukenberg |
leukemia, eosinophilic |
Langerhans Cell Histiocytosis (LCH) |
leukemia, extramedullary |
leiomyoblastoma |
leukemia, hairy cell (HCL) |
leiomyomatosis, intravenous |
leukemia, Hodgkin's cell |
leiomyosarcoma |
leukemia, lymphoblastic, t-cell, acute (ALL) |
leiomyosarcoma, adrenal |
leukemia, prolymphocytic, t-cell |
leiomyosarcoma, epithelioid, gastric leiomyosarcoma, gastric |
leukemia, promyelocytic |
epithelioid |
Leydig cell tumor (LCT) |
leiomyosarcoma, esophagus |
lipoastrocytoma |
leiomyosarcoma, lung |
lipoblastoma |
leiomyosarcoma, oral cavity |
liposarcoma |
leiomyosarcoma, pancreas |
liposarcoma, larynx |
leiomyosarcoma, primary bone (PLMSB) |
liposarcoma, myxoid |
leiomyosarcoma, renal |
liposarcoma, pleomorphic |
leiomyosarcoma, superficial perineal |
liposarcoma, primary mesenteric |
leiomyosarcoma, uterine |
liposarcoma, renal |
leiomyosarcoma, vulva |
liposarcoma, well-differentiated |
leukemia, acute erythroblastic (FAB M6) |
low malignant potential tumor, ovary (LMP) |
leukemia, acute lymphocytic (ALL) |
lymphoepithelioma, parotid gland |
leukemia, acute monocytic |
lymphoma, adrenal |
leukemia, acute myeloid (AML) |
lymphoma, angiocentric |
leukemia, acute nonlymphocytic (ANLL) |
lymphoma, angiotropic large cell |
leukemia, acute nonlymphoblastic |
lymphoma, B-cell |
lymphoma, B-cell, low grade, liver |
lymphoma, large cell, anaplastic |
lymphoma, B-cell, salivary gland |
lymphoma, larynx |
lymphoma, bladder |
lymphoma, lung |
lymphoma, bone |
lymphoma, lymphoblastic (LBL) |
lymphoma, breast |
lymphoma, MALT |
lymphoma, breast, MALT-type |
lymphoma, mantle cell |
lymphoma, Burkitt's |
lymphoma, mediterranean |
lymphoma, cardiovascular system |
lymphoma, muscle |
lymphoma, central nervous system |
lymphoma, nasal |
lymphoma, cervix |
lymphoma, neurological system |
lymphoma, chest wall |
lymphoma, non-Hodgkin's (NHL) |
lymphoma, colorectal mucosa associated lymphoid tumor |
lymphoma, non-Hodgkin's, breast |
lymphoma, cutaneous B cell |
lymphoma, non-Hodgkin's, extranodal localization |
lymphoma, cutaneous T cell (CTCL) |
lymphoma, non-Hodgkin's, larynx |
lymphoma, diffuse large cell |
lymphoma, non-Hodgkin's, pulmonary |
lymphoma, duodenal |
lymphoma, non-Hodgkin's, testis |
lymphoma, endocrine |
lymphoma, ocular |
lymphoma, esophageal |
lymphoma, oral |
lymphoma, follicular |
lymphoma, orbital |
lymphoma, gall bladder |
lymphoma, ovary |
lymphoma, gastrointestinal tract |
lymphoma, pancreatic lymphoma, pancreas |
lymphoma, genital tract |
lymphoma, paranasal sinus |
lymphoma, head & neck |
lymphoma, penile |
lymphoma, heart |
lymphoma, peripheral nervous system |
lymphoma, hepatobilliary |
lymphoma, pharynx |
lymphoma, HIV-associated |
lymphoma, pituitary |
lymphoma, intravascular |
lymphoma, primary breast |
lymphoma, Ki-1 positive, anaplastic, large cell |
lymphoma, primary central nervous system |
lymphoma, kidney |
lymphoma, primary lung |
lymphoma, large bowel |
lymphoma, prostate |
lymphoma, pulmonary |
melanoma, central nervous system |
lymphoma, renal |
melanoma, cervix |
lymphoma, respiratory system |
melanoma, choroidal |
lymphoma, scrotum |
melanoma, conjunctival |
lymphoma, skin |
melanoma, desmoplastic |
lymphoma, small bowel |
melanoma, endocrine |
lymphoma, small intestine |
melanoma, esophageal |
lymphoma, soft tissue |
melanoma, gall bladder |
lymphoma, spermatic cord |
melanoma, gastrointestinal tract |
lymphoma, stomach |
melanoma, genitourinary tract |
lymphoma, t-cell (CTCL) |
melanoma, head & neck |
lymphoma, testicular |
melanoma, heart |
lymphoma, thyroid |
melanoma, intraocular |
lymphoma, trachea |
melanoma, intraoral |
lymphoma, ureter |
melanoma, kidney |
lymphoma, urethra |
melanoma, larynx |
lymphoma, urological system |
melanoma, leptomeningeal |
lymphoma, uterus |
melanoma, lung |
lymphomatosis, intravascular |
melanoma, nasal mucosa |
MALT tumor |
melanoma, oral cavity |
medulloblastoma |
melanoma, osteoid forming/ osteogenic |
melanoma, adrenal |
melanoma, ovary |
melanoma, amelanotic |
melanoma, pancreas |
melanoma, anal |
melanoma, paranasal sinuses |
melanoma, anorectal |
melanoma, parathyroid |
melanoma, biliary tree |
melanoma, penis |
melanoma, bladder |
melanoma, pericardium |
melanoma, brain |
melanoma, pituitary |
melanoma, breast |
melanoma, placenta |
melanoma, cardiopulmonary system |
melanoma, prostate |
melanoma, pulmonary |
mixed mesodermal tumor (MMT) |
melanoma, rectum |
mucosa-associated lymphoid tissue (MALT) |
melanoma, renal pelvis |
Mullerian tumor, malignant mixed, fallopian tube |
melanoma, sinonasal |
Mullerian tumor, malignant mixed, uterine cervix |
melanoma, skeletal system |
myeloma, IgM |
melanoma, small bowel |
myoepithelioma |
melanoma, small intestine |
myoepithelioma, malignant, salivary gland |
melanoma, spinal cord |
nephroblastoma |
melanoma, spleen |
neuroblastoma |
melanoma, stomach |
neuroectodermal tumor, renal |
melanoma, testis |
neuroendocrine tumor, prostate |
melanoma, thyroid |
neurofibrosarcoma |
melanoma, ureter |
nodular hidradenoma, malignant |
melanoma, urethra |
oligodendroglioma |
melanoma, uterus |
oligodendroglioma, anaplastic |
melanoma, vagina |
oligodendroglioma, low-grade |
melanoma, vulva |
osteosarcoma |
meningioma, malignant, anaplastic |
Paget's disease, extramammary (EMPD) |
meningioma, malignant, angioblastic |
Paget's disease, mammary |
meningioma, malignant, atypical |
pancreatoblastoma |
meningioma, malignant, papillary |
paraganglioma, malignant |
mesenchymal neoplasm, stromal |
paraganglioma, malignant, extra-adrenal |
mesenchymoma |
paraganglioma, malignant, gangliocytic |
mesoblastic nephroma |
paraganglioma, malignant, laryngeal |
mesothelioma, malignant |
peripherial nerve sheath tumor, malignant (MPNST) |
mesothelioma, malignant, pleura |
pheochromocytoma, malignant |
mesothelioma, papillary |
phyllodes tumor, malignant, breast |
mesothelioma/tunica vaginalis, malignant (MMTV) |
pilomatrixoma, malignant |
microadenocarcinoma, pancreatic |
plasmacytoma, extramedullary (EMP) |
mixed cell tumor, pancreatic |
plasmacytoma, laryngeal |
plasmacytoma, solitary |
sarcoma, bladder |
pleomorphic adenoma, malignant |
sarcoma, botryoides |
pleomorphic xanthoastrocytoma (PXA) |
sarcoma, central nervous system |
plexiform fibrohistiocytic tumor |
sarcoma, clear cell, kidney |
polyembryoma |
sarcoma, clear cell, soft parts |
polypoid glottic tumor |
sarcoma, dendritic cell, follicular |
primary lesions, malignant, diaphragm |
sarcoma, endometrial stromal (ESS) |
primary malignant lesions, chest wall |
sarcoma, epithelioid |
primary malignant lesions, pleura |
sarcoma, Ewing's (EWS) |
primary sinonasal nasopharyngeal undifferentiated (PSNPC) |
sarcoma, Ewing's, extraosseus (EOE) |
primitive neuroectodermal tumor (PNET) |
sarcoma, Ewing's, primitive neuroectodermal tumor |
proliferating trichilemmal tumor, malignant |
sarcoma, fallopian tube |
pseudomyxoma peritonei, malignant (PMP) |
sarcoma, fibromyxoid |
raniopharyngioma |
sarcoma, granulocytic |
reticuloendothelial tumor |
sarcoma, interdigitating reticulum cell |
retiforme hemangioendothelioma |
sarcoma, intracerebral |
retinoblastoma |
sarcoma, intracranial |
retinoblastoma, trilateral |
sarcoma, Kaposi's |
rhabdoid teratoma, atypical teratoid AT/RT |
sarcoma, Kaposi's, intraoral |
rhabdoid tumor, malignant |
sarcoma, kidney |
rhabdomyosarcoma (RMS) |
sarcoma, mediastinum |
rhabdomyosarcoma, orbital |
sarcoma, meningeal |
rhabdomyosarcoma, alveolar |
sarcoma, neurogenic |
rhabdomyosarcoma, botryoid |
sarcoma, ovarian |
rhabdomyosarcoma, central nervous system |
sarcoma, pituitary |
rhabdomyosarcoma, chest wall |
sarcoma, pleomorphic soft tissue |
rhabdomyosarcoma, paratesticular (PTR) |
sarcoma, primary, lung |
sarcoma, adult prostate gland |
sarcoma, primary, pulmonar (PPS) |
sarcoma, adult soft tissue |
sarcoma, prostate |
sarcoma, alveolar soft part (ASPS) |
sarcoma, pulmonary arterial tree |
sarcoma, renal |
stromal cell tumor, sex cord |
sarcoma, respiratory tree |
stromal cell, testicular |
sarcoma, soft tissue |
stromal luteoma |
sarcoma, stromal, gastrointestinal (GIST) |
stromal myosis, endolymphatic (ESM) |
sarcoma, stromal, ovarian |
stromal tumor, colorectal |
sarcoma, synovial |
stromal tumor, gastrointestinal (GIST) |
sarcoma, synovial, intraarticular |
stromal tumor, gonadal (sex cord) (GSTS) |
sarcoma, synovial, lung |
stromal tumor, ovary |
sarcoma, true |
stromal tumor, small bowel |
sarcoma, uterine |
struma ovarii |
sarcoma, vaginal |
teratocarcinosarcoma, sinonasal (SNTCS) |
sarcoma, vulvar |
teratoma, immature |
sarcomatosis, meningeal |
teratoma, intramedullary spine |
sarcomatous metaplasia |
teratoma, mature |
schwannoma, malignant |
teratoma, pericardium |
schwannoma, malignant, cellular, skin |
teratoma, thyroid gland |
schwannoma, malignant, epithelioid |
thecoma stromal luteoma |
schwannoma, malignant, esophagus |
thymoma, malignant |
schwannoma, malignant, nos |
thymoma, malignant, medullary |
Sertoli cell tumor, large cell, calcifying |
thyroid/brain, anaplastic |
sertoli-Leydig cell tumor (SLCT) |
trichoblastoma, skin |
small cell cancer, lungsmall cell lung cancer (SCLC) |
triton tumor, malignant, nasal cavity |
solid-pseudopapillary tumor, pancreas |
trophoblastic tumor, fallopian tube |
somatostinoma |
trophoblastic tumor, placental site |
spindle cell tumor |
urethral cancer |
spindle epithelial tumour w/thymus-like element |
vipoma (islet cell) |
spiradenocylindroma, kidney |
vulvar cancer |
squamous neoplasm, papillary |
Waldenstrom's macroglobullinemia |
steroid cell tumor |
Wilms' tumor Nephroblastoma |
Stewart-Treves syndrome |
Wilms' tumor, lung |
TABLE 3 - Exemplary Cancer Medications
|
Cabazitaxel |
|
Campath (Alemtuzumab) |
Abiraterone Acetate |
Camptosar (Irinotecan Hydrochloride) |
Abitrexate (Methotrexate) |
Capecitabine |
Adriamycin (Doxorubicin Hydrochloride) |
Carboplatin |
Adrucil (Fluorouracil) |
Cerubidine (Daunorubicin Hydrochloride) |
Afinitor (Everolimus) |
Cervarix (Recombinant HPV Bivalent Vaccine) |
Aldara (Imiquimod) |
Cetuximab |
Aldesleukin |
Chlorambucil |
Alemtuzumab |
Cisplatin |
Alimta (Pemetrexed Disodium) |
Clafen (Cyclophosphamide) |
Aloxi (Palonosetron Hydrochloride) |
Clofarabine |
Ambochlorin (Chlorambucil) |
Clofarex (Clofarabine) |
Amboclorin (Chlorambucil) |
Clolar (Clofarabine) |
Aminolevulinic Acid |
Cyclophosphamide |
Anastrozole |
Cyfos (Ifosfamide) |
Aprepitant |
Cytarabine |
Arimidex (Anastrozole) |
Cytarabine, Liposomal |
Aromasin (Exemestane) |
Cytosar-U (Cytarabine) |
Arranon (Nelarabine) |
Cytoxan (Cyclophosphamide) |
Arsenic Trioxide |
Dacarbazine |
Arzerra (Ofatumumab) |
Dacogen (Decitabine) |
Avastin (Bevacizumab) |
Dasatinib |
Azacitidine |
Daunorubicin Hydrochloride |
Bendamustine Hydrochloride |
Decitabine |
Bevacizumab |
Degarelix |
Bexarotene |
Denileukin Diftitox |
Bexxar (Tositumomab and I 131 Iodine Tositumomab) |
Denosumab |
Bleomycin |
DepoCyt (Liposomal Cytarabine) |
Bortezomib |
DepoFoam (Liposomal Cytarabine) |
Dexrazoxane Hydrochloride |
Fulvestrant |
Docetaxel |
Gardasil (Recombinant HPV Quadrivalent Vaccine) |
Doxorubicin Hydrochloride |
Gefitinib |
Efudex (Fluorouracil) |
Gemcitabine Hydrochloride |
Elitek (Rasburicase) |
Gemtuzumab Ozogamicin |
Ellence (Epirubicin Hydrochloride) |
Gemzar (Gemcitabine Hydrochloride) |
Eloxatin (Oxaliplatin) |
Gleevec (Imatinib Mesylate) |
Eltrombopag Olamine |
Halaven (Eribulin Mesylate) |
Emend (Aprepitant) |
Herceptin (Trastuzumab) |
Epirubicin Hydrochloride |
HPV Bivalent Vaccine, Recombinant |
Erbitux (Cetuximab) |
HPV Quadrivalent Vaccine, Recombinant |
Eribulin Mesylate |
Hycamtin (Topotecan Hydrochloride) |
Erlotinib Hydrochloride |
Ibritumomab Tiuxetan |
Etopophos (Etoposide Phosphate) |
Ifex (Ifosfamide) |
Etoposide |
Ifosfamide |
Etoposide Phosphate |
Ifosfamidum (Ifosfamide) |
Everolimus |
Imatinib Mesylate |
Evista (Raloxifene Hydrochloride) |
Imiquimod |
Exemestane |
Ipilimumab |
Fareston (Toremifene) |
Iressa (Gefitinib) |
Faslodex (Fulvestrant) |
Irinotecan Hydrochloride |
Femara (Letrozole) |
Istodax (Romidepsin) |
Filgrastim |
Ixabepilone |
Fludara (Fludarabine Phosphate) |
Ixempra (Ixabepilone) |
Fludarabine Phosphate |
Jevtana (Cabazitaxel) |
Fluoroplex (Fluorouracil) |
Keoxifene (Raloxifene Hydrochloride) |
Fluorouracil |
Kepivance (Palifermin) |
Folex (Methotrexate) |
Lapatinib Ditosylate |
Folex PFS (Methotrexate) |
Lenalidomide |
Folotyn (Pralatrexate) |
Letrozole |
Leucovorin Calcium |
Ofatumumab |
Leukeran (Chlorambucil) |
Oncaspar (Pegaspargase) |
Leuprolide Acetate |
Ontak (Denileukin Diftitox) |
Levulan (Aminolevulinic Acid) |
Oxaliplatin |
Linfolizin (Chlorambucil) |
Paclitaxel |
LipoDox (Doxorubicin Hydrochloride Liposome) |
Palifermin |
Liposomal Cytarabine |
Palonosetron Hydrochloride |
Lupron (Leuprolide Acetate) |
Panitumumab |
Lupron Depot (Leuprolide Acetate) |
Paraplat (Carboplatin) |
Lupron Depot-Ped (Leuprolide Acetate) |
Paraplatin (Carboplatin) |
Lupron Depot-3 Month (Leuprolide Acetate) |
Pazopanib Hydrochloride |
Lupron Depot-4 Month (Leuprolide Acetate) |
Pegaspargase |
Matulane (Procarbazine Hydrochloride) |
Pemetrexed Disodium |
Methazolastone (Temozolomide) |
Platinol (Cisplatin) |
Methotrexate |
Platinol-AQ (Cisplatin) |
Methotrexate LPF (Methotrexate) |
Plerixafor |
Mexate (Methotrexate) |
Pralatrexate |
Mexate-AQ (Methotrexate) |
Prednisone |
Mozobil (Plerixafor) |
Procarbazine Hydrochloride |
Mylosar (Azacitidine) |
Proleukin (Aldesleukin) |
Mylotarg (Gemtuzumab Ozogamicin) |
Prolia (Denosumab) |
Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle |
Promacta (Eltrombopag Olamine) |
Formulation) |
Provenge (Sipuleucel-T) |
Nelarabine |
Raloxifene Hydrochloride |
Neosar (Cyclophosphamide) |
Rasburicase |
Neupogen (Filgrastim) |
Recombinant HPV Bivalent Vaccine |
Nexavar (Sorafenib Tosylate) |
Recombinant HPV Quadrivalent Vaccine |
Nilotinib |
Revlimid (Lenalidomide) |
Nolvadex (Tamoxifen Citrate) |
Rheumatrex (Methotrexate) |
Nplate (Romiplostim) |
Rituxan (Rituximab) |
Rituximab |
Tositumomab and I 131 Iodine Tositumomab |
Romidepsin |
Totect (Dexrazoxane Hydrochloride) |
Romiplostim |
Trastuzumab |
Rubidomycin (Daunorubicin Hydrochloride) |
Treanda (Bendamustine Hydrochloride) |
Sclerosol Intrapleural Aerosol (Talc) |
Trisenox (Arsenic Trioxide) |
Sipuleucel-T |
Tykerb (Lapatinib Ditosylate) |
Sorafenib Tosylate |
Vandetanib |
Sprycel (Dasatinib) |
Vectibix (Panitumumab) |
Sterile Talc Powder (Talc) |
Velban (Vinblastine Sulfate) |
Steritalc (Talc) |
Velcade (Bortezomib) |
Sunitinib Malate |
Velsar (Vinblastine Sulfate) |
Sutent (Sunitinib Malate) |
VePesid (Etoposide) |
Synovir (Thalidomide) |
Viadur (Leuprolide Acetate) |
Talc |
Vidaza (Azacitidine) |
Tamoxifen Citrate |
Vinblastine Sulfate |
Tarabine PFS (Cytarabine) |
Vincasar PFS (Vincristine Sulfate) |
Tarceva (Erlotinib Hydrochloride) |
Vincristine Sulfate |
Targretin (Bexarotene) |
Vorinostat |
Tasigna (Nilotinib) |
Votrient (Pazopanib Hydrochloride) |
Taxol (Paclitaxel) |
Wellcovorin (Leucovorin Calcium) |
Taxotere (Docetaxel) |
Xeloda (Capecitabine) |
Temodar (Temozolomide) |
Xgeva (Denosumab) |
Temozolomide |
Yervoy (Ipilimumab) |
Temsirolimus |
Zevalin (Ibritumomab Tiuxetan) |
Thalidomide |
Zinecard (Dexrazoxane Hydrochloride) |
Thalomid (Thalidomide) |
Zoledronic Acid |
Toposar (Etoposide) |
Zolinza (Vorinostat) |
Topotecan Hydrochloride |
Zometa (Zoledronic Acid) |
Toremifene |
Zytiga (Abiraterone Acetate) |
Torisel (Temsirolimus) |
|
TABLE 4 - Exemplary Ocular Diseases and Conditions
|
|
• |
keratoconjunctivitis sicca (dry eye syndrome) |
Examples of "back of the eye" diseases include |
• |
iridocyclitis |
• |
macular edema such as angiographic cystoid macular |
• |
iritis |
|
edema |
• |
scleritis |
• |
retinal ischemia and choroidal neovascularization |
• |
episcleritis |
• |
macular degeneration |
• |
corneal edema |
• |
retinal diseases (e.g., diabetic retinopathy, diabetic |
• |
scleral disease |
|
retinal edema, retinal detachment); inflammatory |
• |
ocular cicatrcial pemphigoid |
|
diseases such as uveitis (including panuveitis) or |
• |
pars planitis |
|
choroiditis (including multifocal choroiditis) of |
• |
Posner Schlossman syndrome |
|
unknown cause (idiopathic) or associated with a |
• |
Behcet's disease |
|
systemic (e.g., autoimmune) disease; episcleritis or |
• |
Vogt-Koyanagi-Harada syndrome |
|
scleritis |
• |
hypersensitivity reactions |
• |
Birdshot retinochoroidopathy |
• |
conjunctival edema |
• |
vascular diseases (retinal ischemia, retinal vasculitis, |
|
choroidal vascular insufficiency, choroidal |
• |
conjunctival venous congestion |
|
thrombosis) |
• |
periorbital cellulitis; acute dacryocystitis |
• |
neovascularization of the optic nerve |
• |
non-specific vasculitis |
• |
optic neuritis |
• |
sarcoidosis |
Examples of "front-of-eye" diseases include: |
|
|
• |
blepharitis |
|
|
• |
keratitis |
|
|
• |
rubeosis iritis |
|
|
• |
Fuchs' heterochromic iridocyclitis |
|
|
• |
chronic uveitis or anterior uveitis |
|
|
• |
conjunctivitis |
|
|
• |
allergic conjunctivitis (including seasonal or perennial, vernal, atopic, and giant
papillary) |
|
|
TABLE 5 -
Exemplary Ocular Medications
Atropine |
Brimondine (Alphagan) |
Ciloxan |
Erythromycin |
Gentamicin |
Levobunolol (Betagan) |
Metipranolol (Optipranolol) |
Optivar |
Patanol |
PredForte |
Proparacaine |
Timoptic |
Trusopt |
Visudyne (Verteporfin) |
Voltaren |
Xalatan |
TABLE 6 - Exemplary Diseases and Conditions affecting the Lungs
Acute Bronchitis |
Pulmonary Arterial Hypertension |
Acute Respiratory Distress Syndrome (ARDS) |
Pulmonary Fibrosis |
Asbestosis |
Pulmonary Vascular Disease |
Asthma |
Respiratory Syncytial Virus |
Bronchiectasis |
Sarcoidosis |
Bronchiolitis |
Severe Acute Respiratory Syndrome |
Bronchopulmonary Dysplasia |
Silicosis |
Byssinosis |
Sleep Apnea |
Chronic Bronchitis |
Sudden Infant Death Syndrome |
Coccidioidomycosis (Cocci) |
Tuberculosis |
COPD |
|
Cystic Fibrosis |
|
Emphysema |
|
Hantavirus Pulmonary Syndrome |
|
Histoplasmosis |
|
Human Metapneumovirus |
|
Hypersensitivity Pneumonitis |
|
Influenza |
|
Lung Cancer |
|
Lymphangiomatosis |
|
Mesothelioma |
|
Nontuberculosis Mycobacterium |
|
Pertussis |
|
Pneumoconiosis |
|
Pneumonia |
|
Primary Ciliary Dyskinesia |
|
Primary Pulmonary Hypertension |
|
TABLE 7 - Exemplary Lung/Respiratory disease medications:
|
Curosurf |
|
Daliresp (roflumilast) |
Accolate |
Dulera (mometasone furoate + formoterol fumarate dihydrate) |
Accolate |
DuoNeb (albuterol sulfate and ipratropium bromide) |
Adcirca (tadalafil) |
Dynabac |
Aldurazyme (laronidase) |
Flonase Nasal Spray |
Allegra (fexofenadine hydrochloride) |
Flovent Rotadisk |
Allegra-D |
Foradil Aerolizer (formoterol fumarate) inhalation powder) |
Alvesco (ciclesonide) |
Infasurf |
Astelin nasal spray |
Invanz |
Atrovent (ipratropium bromide) |
Iressa (gefitinib) |
Augmentin (amoxicillin/clavulanate) |
Ketek (telithromycin) |
Avelox I.V. (moxifloxacin hydrochloride) |
Letairis (ambrisentan) |
Azmacort (triamcinolone acetonide) Inhalation Aerosol |
Metaprotereol Sulfate Inhalation Solution, 5% |
Biaxin XL (clarithromycin extended-release tablets) |
Nasacort AQ (triamcinolone acetonide) Nasal Spray |
Breathe Right |
Nasacort AQ (triamcinolone acetonide) Nasal Spray |
Brovana (arformoterol tartrate) |
NasalCrom Nasal Spray |
Cafcit Injection |
OcuHist |
Cayston (aztreonam for inhalation solution) |
Omnicef |
Cedax (ceftibuten) |
Patanase (olopatadine hydrochloride) |
Cefazolin and Dextrose USP |
Priftin |
Ceftin (cefuroxime axetil) |
Proventil HFA Inhalation Aerosol |
Cipro (ciprofloxacin HCl) |
Pulmozyme (dornase alfa) |
Clarinex |
Pulmozyme (dornase alfa) |
Claritin RediTabs (10 mg loratadine rapidly-disintegrating tablet) |
Qvar (beclomethasone dipropionate) |
Claritin Syrup (loratadine) |
Raxar (grepafloxacin) |
Claritin-D 24 Hour Extended Release Tablets (10 mg loratadine, |
Remodulin (treprostinil) |
240 mg pseudoephedrine sulfate) |
RespiGam (Respiratory Syncitial Virus Immune Globulin |
Clemastine fumarate syrup |
Intravenous) |
Covera-HS (verapamil) |
Rhinocort Aqua Nasal Spray |
Sclerosol Intrapleural Aerosol |
Zosyn (sterile piperacillin sodium/tazobactam sodium) |
Serevent |
Zyflo (Zileuton) |
Singulair |
Zyrtec (cetirizine HCl) |
Spiriva HandiHaler (tiotropium bromide) |
|
Synagis |
|
Tavist (clemastine fumarate) |
|
Tavist (clemastine fumarate) |
|
Teflaro (ceftaroline fosamil) |
|
Tequin |
|
Tikosyn Capsules |
|
Tilade (nedocromil sodium) |
|
Tilade (nedocromil sodium) |
|
Tilade (nedocromil sodium) |
|
Tobi |
|
Tracleer (bosentan) |
|
Tri-Nasal Spray (triamcinolone acetonide spray) |
|
Tripedia (Diptheria and Tetanus Toxoids and Acellular Pertussis |
|
Vaccine Absorbed) |
|
Tygacil (tigecycline) |
|
Tyvaso (treprostinil) |
|
Vancenase AQ 84 mcg Double Strength |
|
Vanceril 84 mcg Double Strength (beclomethasone dipropionate, 84 |
|
mcg) Inhalation Aerosol |
|
Ventolin HFA (albuterol sulfate inhalation aerosol) |
|
Visipaque (iodixanol) |
|
Xolair (omalizumab) |
|
Xopenex |
|
Xyzal (levocetirizine dihydrochloride) |
|
Zagam (sparfloxacin) tablets |
|
Zemaira (alphal-proteinase inhibitor) |
|
TABLE 8 -
Exemplary Diseases and Conditions affecting the Heart:
Heart attack |
Atherosclerosis |
High blood pressure |
Ischemic heart disease |
Heart rhythm disorders |
Tachycardia |
Heart murmurs |
Rheumatic heart disease |
Pulmonary heart disease |
Hypertensive heart disease |
Valvular heart disease |
Infective endocarditis |
Congenital heart diseases |
Coronary heart disease |
Atrial myxoma |
HOCM |
Long QT syndrome |
Wolff Parkinson White syndrome |
Supraventricular tachycardia |
Atrial flutter |
Constrictive pericarditis |
Atrial myxoma |
Long QT syndrome |
Wolff Parkinson White syndrome |
Supraventricular tachycardia |
Atrial flutter |
TABLE 9 - Exemplary Heart Medications
|
nadolol, Corgard |
|
niacin and lovastatin, Advicor |
ACE Inhibitors |
niacin, Niacor, Niaspan, Slo-Niacin |
acetylsalicylic acid, Aspirin, Ecotrin |
nitroglycerin, Nitro-Bid, Nitro-Dur, Nitrostat, Transderm- |
alteplase, Activase, TPA |
Nitro, Minitran, Deponit, Nitrol |
anistreplase-injection, Eminase |
oxprenolol-oral |
Aspirin and Antiplatelet Medications |
pravastatin, Pravachol |
atenolol, Tenormin |
pravastatin/buffered aspirin-oral, Pravigard PAC |
atorvastatin, Lipitor |
propranolol, Inderal, Inderal LA |
benazepril, Lotensin |
quinapril hcl/hydrochlorothiazide-oral, Accuretic |
Beta Blockers |
quinapril, Accupril |
Bile Acid Sequestrants |
ramipril, Altace |
Calcium Channel Blockers |
reteplase-injection, Retavase |
captopril and hydrochlorothiazide, Capozide |
simvastatin, Zocor |
captopril, Capoten |
Statins |
clopidogrel bisulfate, Plavix |
streptokinase-injection, Kabikinase, Streptase |
colesevelam, Welchol |
torsemide-oral, Demadex |
dipyridamole-oral, Persantine |
trandolapril, Mavik |
enalapril and hydrochlorothiazide, Vaseretic |
|
enalapril, Vasotec |
|
ezetimibe and simvastatin, Vytorin |
|
Fibrates |
|
fluvastatin, Lescol |
|
fosinopril sodium, Monopril |
|
lisinopril and hydrochlorothiazide, Zestoretic, Prinzide |
|
lisinopril, Zestril, Prinivil |
|
lovastatin, Mevacor, Altocor |
|
magnesium sulfate-injection |
|
metoprolol, Lopressor, Toprol XL |
|
moexipril-oral, Univasc |
|
TABLE 10 -
Exemplary Bacterial, Viral, Fungal and Parasitic Conditions
Bacterial Infections caused by: |
• |
Borrelia species |
• |
Streptococcus pneumoniae |
• |
Staphylococcus aureus |
• |
Mycobacterium tuberculosis |
• |
Mycobacterium leprae |
• |
Neisseria gonorrheae |
• |
Chlamydia trachomatis |
• |
Pseudomonas aeruginosa |
|
Viral Infections caused by: |
• |
Herpes simplex |
• |
Herpes zoster |
• |
cytomegalovirus |
|
Fungal Infections caused by: |
• |
Aspergillus fumigatus |
• |
Candida albicans |
• |
Histoplasmosis capsulatum |
• |
Cryptococcus species |
• |
Pneumocystis carinii |
|
Parasitic Infections caused by: |
• |
Toxoplasmosis gondii |
• |
Trypanosome cruzi |
• |
Leishmania species |
• |
Acanthamoeba species |
• |
Giardia lamblia |
• |
Septata species |
• |
Dirofilaria immitis |